A2-73 crystalline polymorph compositions of matter and methods of use thereof

ABSTRACT

The present disclosure provides crystalline forms of tetrahydro-N,N-dimethyl-2,2-diphenyl-3-furanmethanamine (A2-73), in freebase or salt forms. Also described are pharmaceutical formulations and dosage forms comprising the disclosed crystal forms, and methods of using crystalline A2-73 in dosage forms for neuroprotection including treatment of neurodegenerative and other diseases.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication No. 62/656,435 filed Apr. 12, 2018, the entire disclosure ofwhich is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates generally to crystalline forms oftetrahydro-N,N-dimethyl-2,2-diphenyl-3-furanmethanamine (A2-73), dosageforms containing them and methods of their use in treatment.

BACKGROUND OF THE INVENTION

Tetrahydro-N,N-dimethyl-2,2-diphenyl-3-furanmethanamine (ANAVEX2-73 orAV2-73) is a mixed muscarinic receptor ligand and Sig-1R agonist withaffinities in the low micromolar range. A2-73 can treatneurodevelopmental disorders and neuroprotective characteristics.Improved drug formulations showing, for example, better bioavailability,better stability, or enhanced delivery of pharmaceutically activecompounds are consistently sought, there is an ongoing need for morefully characterized, new, drug molecules. There is also an ongoing needfor methods of treating neurodegenerative diseases.

SUMMARY OF THE INVENTION

In one aspect, the present disclosure encompasses a crystalline form oftetrahydro-N,N-dimethyl-2,2-diphenyl-3-furanmethanamine (A2-73), whereinthe crystalline form is a salt or a freebase. A salt can be anypharmaceutically acceptable salt, such as a hydrochloride salt, afumarate salt, a sulfate salt, a dihydrogen phosphate salt, a benzoatesalt, a mesylate salt, an edisylate salt, and an oxalate salt. It shallbe understood that in any of the pharmaceutical formulations, dosageforms and methods disclosed herein, crystalline A2-73 can be thefreebase disclosed herein, or a salt as disclosed herein including anyone or more of a hydrochloride salt, a fumarate salt, a sulfate salt, adihydrogen phosphate salt, a benzoate salt, a mesylate salt, anedisylate salt, and an oxalate salt.

When the A2-73 is a hydrochloride salt, the hydrochloride salt ischaracterized by the XRPD pattern shown in FIG. 4, FIG. 6, FIG. 8, FIG.9, FIG. 10, FIG. 11, FIG. 12, and FIG. 14. The hydrochloride saltcharacterized by the XRPD pattern shown in FIG. 4 can be furthercharacterized by the particle shapes and sizes depicted in FIG. 2 andFIG. 3. The hydrochloride salt characterized by the XRPD pattern shownin FIG. 6 can be further characterized by the particle shapes and sizesdepicted in FIG. 5. hydrochloride salt characterized by the XRPD patternshown in FIG. 8 can be further characterized by the particle shapes andsizes depicted in FIG. 7. The hydrochloride salt characterized by theXRPD pattern shown in FIG. 14 is further characterized by the particleshapes and sizes depicted in FIG. 13.

The crystalline form of A2-73 can be a sulfate salt. The sulfate saltcan be characterized by the XRPD pattern shown in FIG. 18 and FIG. 19.The sulfate salt characterized by the XRPD pattern shown in FIG. 18 canbe further characterized by the particle shapes depicted in FIG. 17.

The crystalline form of A2-73 can be a mesylate salt. The mesylate saltcan be characterized by the XRPD pattern shown in FIG. 20.

The crystalline form of A2-73 can be an oxalate salt. The oxalate saltcan be characterized by the XRPD pattern shown in FIG. 21, FIG. 22, andFIG. and 23.

The crystalline form of A2-73 can be a dihydrogen phosphate salt. Thedihydrogen phosphate salt can be characterized by the XRPD pattern shownin FIG. 25. The dihydrogen phosphate salt characterized by the XRPDpattern shown in FIG. 25 is further characterized by the particle shapesdepicted in FIG. 24.

The crystalline form of A2-73 can be an edisylate salt. The edisylatesalt can be characterized by the XRPD pattern shown in FIG. 26.

The crystalline form of A2-73 can be a benzoate salt. The benzoate saltcan be characterized by the XRPD pattern shown in FIG. 27.

The crystalline form of A2-73 can be a fumarate salt. The fumarate saltcan be characterized by the XRPD pattern shown in FIG. 29, FIG. 30, FIG.32, FIG. 33, and FIG. 34. The fumarate salt characterized by the XRPDpattern shown in FIG. 29 can be further characterized by the particleshapes depicted in FIG. 28, and the fumarate salt characterized by theXRPD pattern shown in FIG. 32 can be further characterized by theparticle shapes depicted in FIG. 31.

The crystalline form of A2-73 can be a freebase. The freebase can becharacterized by the XRPD pattern shown in FIG. 16. The crystalline formcharacterized by the XRPD pattern shown in FIG. 16 is furthercharacterized by the particle shapes depicted in FIG. 15.

In another aspect, the present disclosure encompasses a dosage formcomprising a therapeutically effective amount of A2-73 in a crystallineform selected from the group consisting of A2-73 freebase and a A2-73salt. The dosage form can comprise from about 1 mg to about 50 g, fromabout 1 mg to about 500 mg, or about 1 mg to about 100 mg of A2-73freebase or an A2-73 salt.

A dosage form can be formulated for extended release of crystallineA2-73. In any dosage form, crystalline A2-73 can be a freebase, and thedosage form can comprise from about 1 mg to about 500 mg of A2-73freebase. A dosage form can be a transdermal patch. A transdermal patchcan contain from about 40 mg to about 60 mg, from about 80 mg to about120 mg, or about 180 mg to about 220 mg of A2-73 freebase. The dosageform can be an enteric coated oral formulation, and the formulation cancomprise from about 1 mg to about 50 mg A2-73 freebase.

In any dosage form, crystalline A2-73 can be a pharmaceuticallyacceptable salt. A pharmaceutically acceptable salt can be selected fromthe group consisting of fumarate, sulfate, mesylate, dihydrogenphosphate, edisylate, benzoate, hydrochloride, and oxalate. In someaspects, an A2-73 salt is fumarate, and the dosage form can be atransdermal patch. The transdermal patch can contain from about 1 mg toabout 55 mg of A2-73 fumarate salt.

In some aspects, a dosage form can be an enteric coated oralformulation. The enteric coated oral formulation can comprise from about10 mg to about 50 mg, from about 20 mg to about 30 mg, or from about 15mg to about 25 mg of A2-73 fumarate salt.

In another aspect, the present disclosure encompasses a pharmaceuticalformulation for delivery of A2-73. The formulation comprises atherapeutically effective amount of a crystalline form of A2-73 selectedfrom A2-73 freebase and A2-73 salt.

A pharmaceutical formulation can further comprise one or morepharmaceutically acceptable excipients selected from chemical enhancers,humectants, pressure sensitive adhesives, antioxidants, solubilizers,thickening agents, plasticizers, adjuvants, carriers, excipients,vehicles, and any combinations thereof. The one or more excipients canbe selected for preparing the formulation for oral, transdermal,parenteral, intraperitoneal, intravascular, subcutaneous, by inhalationspray, rectal, or intrapulmonary administration.

In any pharmaceutical formulation, crystalline A2-73 can be selectedfrom freebase, and any pharmaceutically acceptable salt. In one aspectof a pharmaceutical formulation, crystalline A2-73 is a fumarate salt,or a hydrochloride salt. A pharmaceutical formulation can be for examplean oral formulation comprising from about 1% to about 100% by weightcrystalline A2-73.

A pharmaceutical formulation can be prepared for extended delivery ofcrystalline A2-73 and can comprise from about 1 mg to about 50 g ofcrystalline A2-73. An extended delivery formulation can for example be asubcutaneous injectable dosage formulation comprising from about 0.5 gto about 3 g of crystalline A2-73.

A pharmaceutical formulation can be a transdermal patch. The patch cancomprise from about 40 mg to about 60 mg, from about 80 mg to about 120mg, or about 180 mg to about 220 mg of A2-73 freebase. The patch canalso comprise from about 1 mg to about 55 mg of A2-73 fumarate salt.

The formulation can also be an oral formulation. The oral formulationcan comprise from about 1 mg to about 50 mg A2-73 freebase, from about10 mg to about 50 mg, from about 20 mg to about 30 mg, or from about 15mg to about 25 mg of A2-73 fumarate salt. The oral formulation cancomprise comprises A2-73 hydrochloride salt.

The extended delivery formulation can be a subcutaneous dosage formcomprising from about 0.1 to about 5 g of crystalline A2-73.

In yet another aspect, the present disclosure encompasses a transdermalpatch for extended delivery of A2-73. The patch may comprise atherapeutically effective amount of a crystalline form of A2-73 selectedfrom A2-73 freebase and A2-73 salt. The transdermal patch can forexample be a matrix patch. The patch can further comprise one or morecomponents selected from chemical enhancers, humectants, pressuresensitive adhesives, antioxidants, solubilizers, thickening agents,plasticizers, and any combinations thereof. The patch can be covered bya peripheral pressure sensitive adhesive that extends beyond the patchin all directions.

A transdermal patch comprising A2-73 freebase can contain from about 40mg to about 60 mg, from about 80 mg to about 120 mg, or about 180 mg toabout 220 mg of A2-73 freebase.

A transdermal patch can comprise A2-73 fumarate salt. A patch comprisingA2-73 fumarate salt can contain from about 1 mg to about 55 mg of A2-73fumarate salt.

The surface area of a transdermal patch in contact with the skin of asubject can range from about 1 cm² to about 20 cm², from about 3 cm² toabout 5 cm², or from about 8 cm² to about 10 cm². The patch can forexample be configured to provide for extended release of A2-73 over aperiod ranging from about 1 day to about 7 days. Further, the patch canhave a transcutaneous maximum flux of A2-73 from the matrix ranging fromabout 250-350 μg/cm²/h.

In other aspects, the present disclosure encompasses an oral formulationfor extended delivery of A2-73. The oral formulation comprises a corecomprising a therapeutically effective amount of a crystalline form ofA2-73 selected from A2-73 freebase and A2-73 salt; and an entericcoating surrounding the core.

The oral formulation can comprise A2-73 freebase that can range fromabout 1 mg to about 50 mg A2-73 freebase. The oral formulation can alsocomprise A2-73 fumarate salt that can range in the core from about 10 mgto about 50 mg, from about 20 mg to about 30 mg, or from about 15 mg toabout 25 mg of A2-73 fumarate salt, or about 35% to about 40% by weightA2-73 freebase or A2-73 fumarate. The oral formulation can also comprisein the core about 55% to about 70% by weight hydroxypropylmethylcellulose acetate succinate, about 0.3% to about 0.9% by weightmagnesium stearate, and about 0.05% to about 0.5% by weight colloidalsilicon dioxide. The hydroxypropyl methylcellulose acetate succinate canbe soluble in aqueous solutions with a pH of about 5.5 and greater, asecond grade of hydroxypropyl methylcellulose acetate succinate issoluble in aqueous solutions with a pH of about 6.8 and greater, andcombinations thereof. The formulation can provide for extended releaseof A2-73 over a period ranging from about 1 day to about 3 days, and candeliver about 15 to about 30 mg/day of A2-73 to a subject.

In one aspect, the disclosure encompasses a method of administeringA2-73 to a subject in need thereof. The method comprises administeringthe A2-73 to the subject a crystalline form of A2-73 selected from A2-73freebase and a pharmaceutically acceptable salt of A2-73. In variousaspects of the methods, crystalline A2-73 can be administered in adosage form or pharmaceutical formulation comprising crystalline A2-73freebase, or a pharmaceutically acceptable salt of A2-73 as disclosedherein. A dosage form can be an immediate release or an extended releasedosage form as disclosed herein. In certain aspects, the salt can be afumarate salt, or a hydrochloride salt. In other aspects, crystallineA2-73 can be administered to the subject over a period of about 30 days,about 60 days, about 120 days or about 180 days.

In one aspect, administration can comprise administering using anextended release dosage form which can be administered dermally using atransdermal patch. The transdermal patch can be for example be replacedperiodically such as daily, every other day, weekly, every 10 days totwo weeks, or monthly or more. In one aspect, a transdermal patch canmaintain a level of A2-73 in the blood of the subject ranging from about5 ng/ml to about 15 ng/ml, and particularly about 10 ng/ml over theperiod.

In another aspect, administration can comprise administering using anenteric coated oral dosage form comprising crystalline A2-73. Theenteric coated oral dosage form can be administered daily, or everyother day and can deliver about 15 to about 30 mg/day of A2-73. Anenteric coated oral dosage form comprising crystalline A2-73 can provideadministration of A2-73 over an extended period of time which can be forexample from about 1 day, about 2 days (about 48 hours), 3 days (about72 hours), about 4 days, about 5 days, about 6 days, about 7 days, ormore.

In another aspect, the disclosure encompasses a method of treatingAlzheimer's disease in a subject in need thereof, the method comprisingadministering to the subject a dosage form comprising a therapeuticallyeffective amount of a crystalline form of A2-73 selected from A2-73freebase and A2-73 salt.

In another aspect, the disclosure encompasses a method of treating aprogressive dementia in a subject in need thereof, the method comprisingadministering to the subject a dosage form comprising a therapeuticallyeffective amount of a crystalline form of A2-73 selected from A2-73freebase and A2-73 salt.

In any of the methods, the dosage form being administered can be anextended dosage form as described herein.

In another aspect, the disclosure encompasses a pharmaceuticalcomposition for the treatment of a neurodegenerative disease comprisingan anti-neurodegenerative effective amount of A2-73. The therapeuticallyeffective amount can range from about 0.5 mg to about 20 mg, from about1 mg to about 60 mg, from about 30 mg to about 50 mg, or from about 3 mgto about 5 mg.

In another aspect, the disclosure encompasses a dosage form comprisingan anti-neurodegenerative effective amount of A2-73 effective for thetreatment of a neurodegenerative disease. The amount ofanti-neurodegenerative effective amount of A2-73 can be from about 0.01to about 10 mg/kg or from about 0.01 to about 10 mg/kg.

In one aspect, the disclosure encompasses a method of treating aneurodegenerative disease in a subject in need thereof. The methodcomprises administering to the subject an anti-neurodegenerativeeffective amount of A2-73. The degenerative disease can be Alzheimer'sdisease, Parkinson's disease, prion diseases, Huntington's disease,motor neuron diseases (MND) such as amyotrophic lateral sclerosis,spinocerebellar ataxia (SCA), or spinal muscular atrophy (SMA).

The anti-neurodegenerative effective amount of A2-73 may be about 0.5mg/day to about 100 mg/day, about 1 to about 60 mg/day, about 20 toabout 50 mg/day, about 20 to about 30 mg/day, or about 15 to about 25mg/day. Further, administering to the subject an anti-neurodegenerativeeffective amount of A2-73 can provide blood levels of A2-73 of about 10ng/ml, or about 12 ng/ml.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is an overlay of XRPD patterns for Anavex2-73 (hydrochloridesalt) solid-state forms.

FIG. 2 shows a micrograph of Form I crystals in polarized-light.

FIG. 3 is a micrograph of crystals of Form I Obtained by Sublimation.

FIG. 4 shows XRPD pattern of Anavex2-73 Form I derived fromsingle-crystal results for copper Kα radiation vs. experimentallymeasured XRPD pattern obtained for an isolated bulk sample of Form I.

FIG. 5 Polarized-light Microscopy (PLM) of Form II.

FIG. 6 shows Form II XRPD Pattern obtained using copper Kα radiation vs.single-crystal derived XRPD pattern.

FIG. 7 Polarized-light Microscopy (PLM) of Form III.

FIG. 8 shows Form III XRPD Pattern obtained using copper Kα radiationvs. single-crystal derived XRPD pattern.

FIG. 9 shows Form IV XRPD Pattern obtained using copper Kα radiation.

FIG. 10 shows Form V XRPD Pattern obtained using copper Kα radiation.

FIG. 11 shows Form VI XRPD Pattern obtained using copper Kα radiation.

FIG. 12 shows Form VII XRPD Pattern obtained using copper Kα radiation.

FIG. 13 Polarized-light Microscopy (PLM) of Form VIII.

FIG. 14 shows Form VIII XRPD Pattern obtained using copper Kα radiationvs. single-crystal derived XRPD pattern.

FIG. 15 Non-Polarized-light Microscopy (PLM) of Freebase Form I.

FIG. 16 XRPD pattern of Anavex2-73 freebase Form I obtained using copperKα radiation.

FIG. 17 Polarized-light Microscopy (PLM) of Anavex2-73 sulfate Form I.

FIG. 18 XRPD pattern of Anavex2-73 sulfate Form I obtained using copperKα radiation.

FIG. 19 Anavex2-73 Sulfate Form II XRPD Pattern.

FIG. 20 Anavex2-73 Mesylate Form I XRPD Pattern.

FIG. 21 Anavex2-73 Oxalate Form I XRPD Pattern.

FIG. 22 Anavex2-73 Oxalate Form II XRPD Pattern.

FIG. 23 Anavex2-73 Oxalate Form III XRPD Pattern.

FIG. 24 Polarized-light Microscopy (PLM) of Anavex2-73 dihydrogenphosphate Form I.

FIG. 25 XRPD pattern of Anavex2-73 dihydrogen phosphate Form I obtainedusing copper Kα radiation.

FIG. 26 A2-73 edisylate Form I XRPD pattern, obtained using copper Kαradiation.

FIG. 27 A2-73 benzoate Form I XRPD pattern, obtained using copper Kαradiation.

FIG. 28 Polarized-light Microscopy (PLM) of Anavex2-73 hydrogen fumarateForm I.

FIG. 29 A2-73 hydrogen fumarate Form I XRPD pattern, obtained usingcopper Kα radiation.

FIG. 30 A2-73 hydrogen fumarate Form II XRPD pattern, obtained usingcopper Kα radiation.

FIG. 31 Polarized-light Microscopy (PLM) of Anavex2-73 hydrogen fumarateForm III.

FIG. 32 XRPD pattern of Anavex2-73 hydrogen fumarate Form III obtainedusing copper Kα radiation.

FIG. 33 XRPD pattern of Anavex2-73 hydrogen fumarate Form IV obtainedusing copper Kα radiation.

FIG. 34 XRPD pattern of Anavex2-73 hydrogen fumarate Form V obtainedusing copper Kα radiation.

FIG. 35 Sig-1R activation enhances autophagic activity. FIG. 35A showsWestern blots and a plot quantifying the results of the Western blots ofthe autophagic flux upon addition of ANAVEX2-73. FIG. 35B are Westernblots and a plot quantifying the results of the Western blots of theautophagic flux upon addition of PRE-084. Statistics are depicted asmean+/−SD. *** p<0.001, ** p<0.01, t-test, n=4. FIG. 35C depictsrepresentative confocal fluorescence microscopic images and plotsquantifying the puncta in HEK293 cells stably transfected with aGFP-LC3B reporter construct (Scale bar=20 μm or 10 μm, respectively.Thirty cells per treatment in three independent experiments. ***p<0.001, t-test.

FIG. 36. Sig-1R activation stimulates ULK1 activation and affectsexpression levels of distinct autophagy network factors. FIG. 36A showsWestern blots of ULK1 phosphorylation at serine 555 (pS555) upontreatment of HeLa cells with ANAVEX2-73 and a plot quantifying theresults of the Western blots. Statistics are depicted as mean±SD. **p<0.01, t-test, n=4. FIG. 36B shows Western blots of ULK1phosphorylation at serine 555 (pS555) upon treatment of HeLa cells withPRE-084. Statistics are depicted as mean±SD. *p<0.05, t-test, n=4. FIG.36C is a plot depicting the relative expression levels of autophagynetwork factors analyzed employing the autophagy qPCR array. Theexpression of each gene is depicted in relation to control cells (setto 1) and the threshold for up- or down-regulation is defined as 1.5 and0.67, respectively.

FIG. 37. Sig-1R activation by ANAVEX2-73 enhances autophagy in C.elegans. FIG. 37A shows a Western blot and a plot quantifying theresults of the Western blots of GFP-LGG1 after treatment of worms withANAVEX2-73. Statistics are depicted as mean±SD. * p<0.05, t-test, n=3.FIG. 37B are representative confocal fluorescence microscopic images ofC. elegans treated with ANAVEX2-73 and BafiA₁ or DMSO, and plotsquantifying the number of puncta in the microscopic images. Scale bar=50and 25 gm. Arrowheads indicate autophagosomal structures. Autophagicflux was calculated as indicated, comparing GFP-positive puncta plusBafiA₁ with puncta in the controls *** p<0.001, t-test.

FIG. 38. Sig-1R activation by ANAVEX2-73 increases proteostasis capacityin C. elegans and ameliorates Aβ42-caused paralysis. FIG. 38A showsrepresentative confocal fluorescence microscopic images of ThioflavinS-positive Aβ42 aggregates in head regions of nematodes. Scale bar=50μm. FIG. 38B is a plot of the analyses of Aβ42-induced paralysis.Statistics were conducted using the log-rank test. Three independentexperiments with a total of approx. 70 worms per treatment.Black=control, light grey=50 μM ANAVEX2-73, dark grey=100 !AMANAVEX2-73.

DETAILED DESCRIPTION

The present disclosure is based in part on the surprising discovery thatcrystalline polymorphs oftetrahydro-N,N-dimethyl-2,2-diphenyl-3-furanmethanamine (“A2-73” or“Anavex2-73”) in freebase or pharmaceutically acceptable salt forms aresuitable for oral, transdermal, subcutaneous, or other forms ofadministration, and can be formulated to provide immediate or extendedrelease of A2-74 upon administration. Crystalline polymorphs of A2-73,dosage forms, and formulations comprising crystalline polymorphs ofA2-73 are described below. Methods of using the crystalline polymorphsof A2-73 for treatment are also disclosed, encompassing use of A2-73 forneuroprotection, wherein neuroprotection includes treatment forneurodegenerative diseases.

I. Crystalline Polymorphs

In one aspect the present disclosure presents a crystalline polymorphsof A2-73. Each crystalline polymorph can be in the form of a freebase orcan be in the form of a salt. The crystalline polymorphs arecharacterized by XRPD and other data provided herein. Properties of eachcrystalline polymorph are described below.

a. Form I, Hydrochloride Salt

Form I is anhydrous, crystalline (birefringent plates and platefragments) and shown via single-crystal x-ray analysis to form racemiccrystals, meaning each individual crystal contains both enantiomers.Form I is the thermodynamically-preferred racemic crystal form of thehydrochloride salt and is the current Anavex2-73 active pharmaceuticalingredient (API). FIG. 2 shows a polarized-light microscopy (PLM) ofForm I. FIG. 3 shows crystals of Form I obtained by sublimation.

Single-Crystal X-Ray Analysis Summary:

-   -   Crystal Type: Racemic    -   Space Group: Monoclinic P21/c    -   Unit Cell Parameters:        -   a=14.1623(4) Å α=90.00°        -   b=9.0974(3) Å β=102.103(3)°        -   c=13.4052(4) Å γ=90.00°        -   Volume=1688.73(9) Å 3        -   Z=4, Z′=1        -   Density (ρcalc)=1.250 g/cm3

XRPD pattern of Anavex2-73 Form I derived from single-crystal resultsfor copper Kα radiation vs. experimentally measured XRPD patternobtained for an isolated bulk sample of Form I is shown in FIG. 4.

The twenty most intense XRPD peaks for Anavex2-73 Form I, measured usingcopper Kα radiation are shown in Table 1.

TABLE 1 XRPD peaks for Anavex2-73 Form I Pos. [°2θ] d-spacing [Å] Rel.Int. [%] 11.74 7.537 43 12.71 6.964 46 13.58 6.523 33 14.02 6.315 7016.00 5.538 83 16.54 5.361 26 19.13 4.640 100  20.40 4.354 39 21.474.139 86 21.92 4.055 30 22.10 4.022 50 23.23 3.829 87 23.43 3.796 3723.66 3.761 11 27.00 3.302 46 28.14 3.171 11 29.07 3.072 10 29.73 3.00321 29.79 3.004 17 32.95 2.716 11

b. Form II, Hydrochloride Salt

Form II is a crystalline hydrate (approximately a monohydrate),consisting of columnar (rod-like) crystals. Single-crystal x-rayanalysis shows Form II is a conglomerate, consisting of homochiralcrystals (meaning a physical mixture of optically-pure crystals of theindividual enantiomers). Form II is slightly hygroscopic, andsingle-crystal results, although modeled upon the basis of 0.8 moles ofwater per mole of Anavex2-73, suggest the crystal lattice couldpotentially hold up to 1.75 moles of water per mole of Anavex2-73,making Form II a likely variable hydrate and less desirable from apharmaceutical development perspective. FIG. 5 shows a polarized-lightmicroscopy (PLM) of Form II.

Single-Crystal X-Ray Analysis Summary:

-   -   Crystal Type: Homochiral    -   Space Group: Orthorhombic P2₁2₁2₁    -   Unit Cell Parameters:        -   a=7.10738(5) Å α=90.00°        -   b=14.22620(10) Å β=90.00°        -   c=17.18510(10) Å γ=90.00°        -   Volume=1737.608(16) Å³        -   Z=4, Z′=1        -   Density (ρcalc)=1.173 g/cm³

XRPD pattern of Anavex2-73 Form II derived from single-crystal resultsfor copper Kα radiation vs. experimentally measured XRPD patternobtained for an isolated bulk sample of Form II are shown in FIG. 6.

The twenty most intense XRPD peaks for Anavex2-73 Form II, measuredusing copper Kα radiation are shown in Table 2.

TABLE 2 XRPD peaks for Anavex2-73 Form II Pos. [°2θ] d-spacing [Å] Rel.Int. [%] 7.61 11.622 19 8.23 10.743 34 10.75 8.228 11 12.03 7.359 1912.45 7.107 60 13.59 6.515 26 14.63 6.054 23 16.51 5.371 20 17.05 5.20220 17.36 5.108 66 19.48 4.557 100  19.75 4.496 14 20.47 4.339 50 21.194.192 18 21.67 4.101 37 22.34 3.979 30 23.17 3.838 16 27.93 3.194 3929.97 2.982 17 30.50 2.931 17

c. Form III, Hydrochloride Salt

Form III is a slightly-hygroscopic, anhydrous, crystalline material,exhibiting a columnar (rod-like) morphology. In some aspects,single-crystal x-ray analysis shows Form III is an optically pure form.In other aspects, single-crystal x-ray analysis shows Form III is aconglomerate, consisting of homochiral crystals (meaning a physicalmixture of optically-pure crystals of the individual enantiomers). FormIII appears to be the thermodynamically-preferred optically pure form ofAnavex2-73. FIG. 7 shows polarized-light microscopy (PLM) of Form III.

Single-Crystal X-Ray Analysis Summary:

-   -   Crystal Type: Homochiral    -   Space Group: Orthorhombic P212121    -   Unit Cell Parameters:        -   a=7.10738(5) Å α=90.00°        -   b=14.22620(10) Å β=90.00°        -   c=17.18510(10) Å γ=90.00°        -   Volume=1737.608(16) Å 3        -   Z=4, Z′=1        -   Density (ρcalc)=1.215 g/cm3

XRPD pattern of Anavex2-73 Form III derived from single-crystal resultsfor copper Kα radiation vs. experimentally measured XRPD patternobtained for an isolated bulk sample of Form III (FIG. 8).

The twenty most intense XRPD peaks for Anavex2-73 Form III, measuredusing copper Kα radiation are shown in Table 3.

TABLE 3 XRPD peaks for Anavex2-73 Form III Pos. [°2θ] d-spacing [Å] Rel.Int. [%] 10.14 8.721 27 11.72 7.550 22 11.87 7.455 62 13.30 6.657 9914.67 6.041 20 15.97 5.551 68 17.10 5.185 100  17.43 5.088 29 18.194.877 28 20.23 4.390 50 21.19 4.193 18 23.81 3.734 43 23.88 3.727 3424.56 3.625 30 25.23 3.530 19 26.00 3.427 18 27.49 3.242 26 27.55 3.23724 28.21 3.163 26 29.65 3.013 19

d. Form IV, Hydrochloride Salt

Form IV is crystalline and can be isolated as a physical mixture withamorphous material during lyophilization of Anavex2-73 from water. XRPDpattern of Anavex2-73 Form IV obtained using copper Kα radiation is seenin FIG. 9.

The eleven most intense XRPD peaks for Anavex2-73 Form IV, measuredusing copper Kα radiation are shown in Table 4.

TABLE 4 XRPD peaks for Anavex2-73 Form IV Pos. [°2θ] d-spacing [Å] Rel.Int. [%] 11.92 7.424 56 12.45 7.111 100  13.83 6.403 82 15.57 5.691 6118.65 4.759 35 20.42 4.350 44 21.35 4.161 57 21.94 4.052 70 23.37 3.80737 23.95 3.716 41 26.74 3.334 10

e. Form V, Hydrochloride Salt

Form V is crystalline and can be isolated upon rotary evaporation ofAnavex2-73 from dichloromethane. XRPD pattern of Anavex2-73 Form Vobtained using copper Kα radiation can be seen in FIG. 10. The twentymost intense XRPD peaks for Anavex2-73 Form V, measured using copper Kαradiation are as shown in Table 5.

TABLE 5 Pos. [°2θ] d-spacing [Å] Rel. Int. [%] 8.43 10.494 54 11.747.541 30 12.23 7.235 29 12.99 6.815 28 13.15 6.731 44 13.60 6.513 100 13.82 6.406 42 14.13 6.268 55 15.99 5.544 53 16.50 5.374 28 16.92 5.23928 19.14 4.638 34 20.40 4.354 29 20.61 4.310 29 21.44 4.144 36 21.694.098 39 21.92 4.055 46 23.21 3.833 60 23.40 3.801 36 26.97 3.306 38

f. Form VI, Hydrochloride Salt

Form VI is crystalline and was isolated upon rapid cooling of an aqueoussolution of Anavex2-73 to 5° C. XRPD pattern of Anavex2-73 Form VIobtained using copper Kα radiation is seen in FIG. 11.

The twenty most intense XRPD peaks for Anavex2-73 Form VI, measuredusing copper Kα radiation are as shown in Table 6.

TABLE 6 XRPD peaks for Anavex2-73 Form VI Pos. [°2θ] d-spacing [Å] Rel.Int. [%] 10.72 8.253 8 11.29 7.836 75 11.46 7.723 15 11.79 7.506 3515.85 5.591 13 16.30 5.439 8 17.26 5.138 21 18.45 4.810 32 20.43 4.347100 20.59 4.314 18 22.30 3.986 15 23.55 3.778 14 23.86 3.730 29 25.893.438 70 25.94 3.435 73 26.84 3.322 10 28.65 3.116 13 31.90 2.806 933.60 2.665 11 33.68 2.661 13

g. Form VII, Hydrochloride Salt

Form VII is both crystalline and anhydrous, and it was isolated via airevaporation of Anavex2-73 from methanol. XRPD pattern of Anavex2-73 FormVII obtained using copper Kα radiation is shown in FIG. 12. The twentymost intense XRPD peaks for Anavex2-73 Form VII, measured using copperKα radiation are shown in Table 7.

TABLE 7 XRPD peaks for Anavex2-73 Form VII Pos. [°2θ] d-spacing [Å] Rel.Int. [%] 12.32 7.184 20 12.51 7.076 43 13.91 6.360 25 13.97 6.338 3314.15 6.260 15 14.58 6.076 25 16.61 5.338 78 17.12 5.179 73 17.81 4.98242 20.90 4.251 36 21.77 4.082 16 23.82 3.736 100  24.60 3.619 30 25.213.532 29 25.91 3.439 75 28.95 3.085 19 30.19 2.958 24 30.26 2.953 3030.66 2.914 31 33.99 2.636 21

h. Form VIII, Hydrochloride Salt

Form VIII is a trihydrated, crystalline form of Anavex2-73.Single-crystal x-ray analysis shows Form VIII consists of racemiccrystals, meaning each individual crystal contains both enantiomers.Without being bound by any particular theory, it is believed that FormVIII is a layer or channel hydrate, with the water of hydration beingweakly associated and readily removed by grinding, drying, etc. Theresulting material, a dehydrated lattice, is labile, quickly collapsinginto Form I. FIG. 13 is a polarized-light microscopy (PLM) of Form VIII.

Single-Crystal X-Ray Analysis Summary:

-   -   Crystal Type: Racemic    -   Space Group: Monoclinic P2₁/c    -   Unit Cell Parameters:        -   a=17.7753(11) Å α=90.00°        -   b=9.0306(4) Å β=101.535(5)°        -   c=13.2638(5) Å γ=90.00°        -   Volume=2086.12(12) Å³        -   Z=4, Z′=1        -   Density (ρcalc)=1.184 g/cm³

XRPD pattern of Anavex2-73 Form VIII derived from single-crystal resultsfor copper Kα radiation vs. experimentally measured XRPD patternobtained for an isolated bulk sample of Form VIII is shown in FIG. 14.The twenty most intense XRPD peaks for Anavex2-73 Form VIII, measuredusing copper Kα radiation are shown in Table 8.

TABLE 8 XRPD peaks for Anavex2-73 Form VIII Pos. [°2θ] d-spacing [Å]Rel. Int. [%] 10.13 8.733 28 12.37 7.158 100  13.56 6.532 32 14.09 6.28641 14.70 6.028 18 15.20 5.828 52 16.65 5.325 27 18.18 4.881 61 19.604.530 23 20.31 4.374 70 20.67 4.298 25 21.04 4.222 37 22.12 4.018 1522.64 3.924 96 22.69 3.925 75 23.78 3.738 77 24.92 3.570 23 27.04 3.29523 27.09 3.288 25 29.07 3.069 17

i. Form I, Freebase

A2-73 freebase Form I is crystalline by XRPD and PLM, exhibitinghighly-birefringent agglomerates of columnar (rod-like) crystals.Thermogravimetric analysis (TGA) showed no significant weight loss untilpost-melting, indicating Form I is anhydrous. This is confirmed bygravimetric vapor sorption (GVS) analysis, showing minimal water update(0.3% w/w) up to 90% RH. XRPD analysis post-GVS showed no change inform. Differential scanning calorimetry (DSC) shows a sharp meltingendotherm at an onset of ca. 89° C. (peak temperature at ca. 91° C.).Upon further heating, Form I appears to sublime, beginning above ca.120° C. with a weight loss of ca. 99% observed by 212.6° C. ¹H NMRspectra and HPLC-MS results were consistent with the structure of A2-73freebase. A2-73 freebase was shown to have an HPLC purity of 99.9%, andCAD analysis confirmed the absence of chloride within the sample. FIG.15 is a non-polarized-light microscopy (PLM) of Freebase Form I. XRPDpattern of Anavex2-73 freebase Form I obtained using copper Kα radiationis shown in FIG. 16.

The twenty most intense XRPD peaks for Anavex2-73 freebase Form I,measured using copper Kα radiation are shown in Table 9.

TABLE 9 XRPD peaks for Anavex2-73 freebase Form I Pos. [°2θ] d-spacing[Å] Rel. Int. [%] 10.38 8.519 43 10.82 8.177 15 11.59 7.634 76 12.896.870 15 13.91 6.365 23 15.50 5.717 100  16.24 5.460 70 16.95 5.232 4317.46 5.078 23 17.70 5.012 11 17.98 4.935 37 19.06 4.657 46 20.21 4.39512 20.93 4.244 12 21.27 4.178 29 21.73 4.089 70 22.09 4.023 69 23.533.781 19 24.18 3.681 20 25.71 3.465 13

Solubility:

A2-73 freebase, surprisingly, exhibited high solubility in all solventstested, except water, as shown in Table 10, below.

TABLE 10 Solvent Solubility Screen Results Solvent Approx. Solubility(mg/mL) 1 1,4-Dioxane ≥200 2 1-Butanol ≥200 3 1-Propanol 100 ≥ x ≥ 67  42-Butanone ≥200 5 2-Ethoxyethanol 200 ≥ x ≥ 100 6 2-Propanol 200 ≥ x ≥100 7 Acetone ≥200 8 Acetonitrile 67 ≥ x ≥ 50 9 Cyclohexane ≥200 10Cyclohexanone 200 ≥ x ≥ 100 11 Dichloromethane 200 ≥ x ≥ 100 12 Dimethylsulfoxide ≥200 13 Ethanol 200 ≥ x ≥ 100 14 Ethyl Acetate 200 ≥ x ≥ 10015 Heptane ≥200 16 Isopropyl acetate 67 ≥ x ≥ 50 17 Methanol 200 ≥ x ≥100 18 Methyl Acetate ≥200 19 Methyl isobutyl ketone ≥200 20 N,N′-Dimethylformamide ≥200 21 Nitromethane ≥200 22 t-Butylmethyl ether200 ≥ x ≥ 100 23 Tetrahydrofuran ≥200 24 Water  <5

j. Sulfate Form I

A2-73 sulfate Form I is crystalline and melts at a DSC onset temperatureof ca. 184° C. Form I consisted of highly-birefringent crystals. Its PLMand XRPD pattern are provided below.

FIG. 17 is a polarized-light microscopy (PLM) of Anavex2-73 sulfate FormI. XRPD pattern of Anavex2-73 sulfate Form I obtained using copper Kαradiation is shown in FIG. 18. The twenty most intense XRPD peaks forAnavex2-73 sulfate Form I, measured using copper Kα radiation are shownin Table 11.

TABLE 11 XRPD peaks for Anavex2-73 sulfate Form I Pos. [° 2θ] d-spacing[Å] Rel. Int. [%] 5.88 15.020 21 11.85 7.466 81 13.66 6.483 42 15.675.657 31 15.78 5.612 52 15.84 5.596 69 16.32 5.431 23 17.11 5.181 1418.91 4.692 40 20.30 4.375 23 20.52 4.329 12 20.65 4.302 20 23.82 3.736100 24.56 3.625 18 24.84 3.581 69 24.90 3.581 52 25.80 3.450 12 25.983.426 31 27.03 3.296 12 27.51 3.240 24

k. Sulfate Form II

A2-73 sulfate Form II is crystalline and melts at a DSC onsettemperature of ca. 190° C. Form II appears to be metastable, convertingto A2-73 sulfate Form I post-storage at 40° C./75% RH. Its XRPD pattern,obtained using copper Kα radiation is provided in FIG. 19. The twentymost intense XRPD peaks for Anavex2-73 sulfate Form II, measured usingcopper Kα radiation are shown in Table 12.

TABLE 12 XRPD peaks for Anavex2-73 sulfate Form II Pos. [° 2θ] d-spacing[Å] Rel. Int. [%] 11.50 7.697 20 11.64 7.606 24 12.05 7.347 30 12.117.306 18 13.32 6.647 31 14.73 6.015 18 15.53 5.707 19 15.89 5.577 2619.65 4.514 19 19.75 4.495 33 21.12 4.206 22 23.81 3.737 18 24.00 3.704100 24.07 3.704 60 24.32 3.657 22 25.42 3.501 22 25.60 3.476 57 25.673.476 30 26.42 3.370 45 26.48 3.363 37

I. Mesylate Form I

A2-73 mesylate Form I is crystalline and melts at a DSC onsettemperature of ca. 159° C. Its XRPD pattern, obtained using copper Kαradiation is provided in FIG. 20.

The twenty most intense XRPD peaks for Anavex2-73 mesylate Form I,measured using copper Kα radiation are shown in Table 13.

TABLE 13 XRPD peaks for Anavex2-73 mesylate Form I Pos. [° 2θ] d-spacing[Å] Rel. Int. [%] 11.03 8.019 72 11.15 7.938 99 12.45 7.110 3 13.626.500 36 16.75 5.294 38 17.23 5.145 8 17.98 4.934 48 19.54 4.543 8 20.264.382 3 20.78 4.275 29 22.04 4.030 2 22.13 4.024 2 22.37 3.972 38 22.433.971 17 24.26 3.666 2 24.62 3.614 19 28.51 3.128 100 28.59 3.128 5029.49 3.027 3 33.81 2.649 3

m. Oxalate Form I

A2-73 oxalate Form I is crystalline. Its XRPD pattern, obtained usingcopper Kα radiation is provided in FIG. 21. The twenty most intense XRPDpeaks for Anavex2-73 oxalate Form I, measured using copper Kα radiationare shown in Table 14.

TABLE 14 XRPD peaks for Anavex2-73 oxalate Form I Pos. [° 2θ] d-spacing[Å] Rel. Int. [%] 5.96 14.822 100 6.22 14.212 68 10.18 8.693 35 11.177.924 38 12.68 6.983 31 13.71 6.460 37 14.01 6.320 36 14.68 6.036 4615.63 5.671 32 17.48 5.074 40 18.11 4.899 27 18.34 4.837 36 18.68 4.75065 19.41 4.574 32 19.61 4.528 29 20.20 4.397 41 21.19 4.193 33 21.904.059 28 22.38 3.973 26 23.56 3.776 26

n. Oxalate Form II

A2-73 oxalate Form II is crystalline. Its XRPD pattern, obtained usingcopper Kα radiation is provided in FIG. 22. The twenty most intense XRPDpeaks for Anavex2-73 oxalate Form II, measured using copper Kα radiationin Table 15.

TABLE 15 XRPD peaks for Anavex2-73 oxalate Form II Pos. [° 2θ] d-spacing[Å] Rel. Int. [%] 9.00 9.825 38 9.16 9.657 23 12.76 6.940 56 13.25 6.68319 14.10 6.283 28 14.25 6.217 20 14.64 6.049 28 15.47 5.728 85 15.625.674 75 16.82 5.270 21 18.14 4.891 99 18.36 4.833 23 20.11 4.415 5420.99 4.233 24 22.04 4.033 100 22.73 3.913 38 23.06 3.857 36 24.20 3.67853 24.51 3.632 21 24.70 3.604 21

o. Oxalate Form III

A2-73 oxalate Form III is anhydrous, crystalline and melts with a DSConset temperature of ca. 154° C. Its XRPD pattern, obtained using copperKα radiation is provided in FIG. 23.

The twenty most intense XRPD peaks for Anavex2-73 oxalate Form III,measured using copper Kα radiation.

TABLE 16 Pos. [° 2θ] d-spacing [Å] Rel. Int. [%] 7.50 11.786 30 10.668.302 21 10.86 8.150 82 11.41 7.758 37 12.66 6.991 79 14.20 6.236 10016.09 5.509 57 16.91 5.242 62 17.33 5.118 28 18.17 4.882 27 18.51 4.79473 19.48 4.558 29 20.55 4.322 68 21.17 4.198 31 21.45 4.143 38 23.383.805 25 25.40 3.507 35 25.59 3.481 52 28.41 3.142 19 30.84 2.900 21

p. Dihydrogen Phosphate Form I

A2-73 dihydrogen phosphate (mono-A2-73 phosphate) Form I is crystalline,hygroscopic and melts with a DSC onset temperature of ca. 187-193° C.The isolated sample of Form I consisted of small agglomerates of highlybirefringent crystals, and it exhibits solubilities of ca. 47.2 and 33.1mg/mL at pH 1.2 and 4.5, respectively. The pH of a saturated solution ofForm I in water is 2.66. Its PLM and XRPD pattern are provided in FIGS.24 and 25 respectively.

FIG. 24 is a polarized-light microscopy (PLM) of Anavex2-73 dihydrogenphosphate Form I.

XRPD pattern of Anavex2-73 dihydrogen phosphate Form I obtained usingcopper Kα radiation is shown in FIG. 25.

The twenty most intense XRPD peaks for Anavex2-73 dihydrogen phosphateForm I, measured using copper Kα radiation.

TABLE 17 Pos. [° 2θ] d-spacing [Å] Rel. Int. [%] 5.76 15.334 28 11.697.570 29 13.73 6.450 42 14.36 6.166 19 15.35 5.772 9 16.11 5.502 10017.11 5.184 8 17.32 5.120 9 18.74 4.736 22 20.65 4.301 19 21.14 4.203 2521.88 4.062 7 22.71 3.916 46 23.71 3.752 13 25.34 3.515 12 25.89 3.44215 26.56 3.356 9 26.99 3.304 7 27.66 3.225 14 28.95 3.084 7

q. Edisylate Form I

A2-73 edisylate Form I is crystalline. Its XRPD pattern, obtained usingcopper Kα radiation is provided in FIG. 26.

The twenty most intense XRPD peaks for Anavex2-73 edisylate Form I,measured using copper Kα radiation.

TABLE 16 Pos. [° 2θ] d-spacing [Å] Rel. Int. [%] 3.43 25.781 4 12.916.856 9 13.82 6.406 53 13.96 6.343 15 15.56 5.696 30 16.29 5.443 8 18.604.771 5 18.91 4.692 9 19.20 4.622 25 20.86 4.259 5 21.35 4.162 7 22.333.981 50 24.15 3.685 11 25.49 3.491 62 25.59 3.481 100 29.02 3.077 2229.31 3.047 13 29.52 3.026 16 29.75 3.003 5 33.52 2.674 7

r. Benzoate Form I

A2-73 benzoate Form I is crystalline and melts with a DSC onsettemperature of ca. 116° C. Its XRPD pattern, obtained using copper Kαradiation is provided in FIG. 27.

The twenty most intense XRPD peaks for Anavex2-73 benzoate Form I,measured using copper Kα radiation.

TABLE 17 Pos. [° 2θ] d-spacing [Å] Rel. Int. [%] 10.57 8.373 89 10.738.246 22 11.04 8.018 9 12.30 7.195 19 12.82 6.906 8 15.48 5.725 8 16.025.534 10 16.93 5.237 11 17.31 5.123 100 17.73 5.003 11 19.24 4.614 1519.91 4.459 34 20.56 4.319 9 21.04 4.223 47 21.18 4.194 26 22.10 4.023 824.57 3.623 38 24.76 3.596 17 25.73 3.463 12 32.35 2.767 30

s. Hydrogen Fumarate Form I

A2-73 hydrogen fumarate (mono-A2-73 fumarate) Form I is anhydrous,crystalline and melts with a DSC onset temperature of ca. 193° C. Form Iconsisted of small agglomerates highly-birefringent crystals. Its PLMand XRPD pattern are provided in FIGS. 28 and 29 respectively.

FIG. 28 shows a polarized-light microscopy (PLM) of Anavex2-73 hydrogenfumarate Form I.

XRPD pattern of Anavex2-73 hydrogen fumarate Form I obtained usingcopper Kα radiation I shown in FIG. 29.

The twenty most intense XRPD peaks for Anavex2-73 hydrogen fumarate FormI, measured using copper Kα radiation.

TABLE Pos. [° 2θ] d-spacing [Å] Rel. Int. [%] 10.71 8.264 65 11.94 7.415100 12.17 7.274 29 13.74 6.444 27 14.54 6.092 47 14.85 5.965 56 15.795.613 32 15.93 5.562 60 18.99 4.674 96 20.61 4.309 25 21.02 4.227 3121.51 4.132 26 22.58 3.938 16 23.08 3.853 42 23.29 3.820 34 23.74 3.74830 23.88 3.723 49 23.98 3.717 37 24.86 3.578 19 27.74 3.214 14

t. Hydrogen Fumarate Form II

A2-73 fumarate Form II is crystalline and melts with a DSC onsettemperature of ca. 196° C. Its XRPD pattern, obtained using copper Kαradiation is provided in FIG. 30.

The seventeen most intense XRPD peaks for Anavex2-73 fumarate Form II,measured using copper Kα radiation.

TABLE Pos. [° 2θ] d-spacing [Å] Rel. Int. [%] 11.99 7.382 5.4 14.566.084 100.0 14.86 5.962 2.2 15.78 5.617 0.8 18.99 4.675 19.0 20.69 4.2931.9 22.63 3.929 0.3 23.33 3.813 0.4 23.85 3.731 3.9 24.07 3.695 15.324.13 3.694 7.7 25.70 3.464 0.8 27.80 3.206 0.4 29.31 3.045 7.7 29.393.045 4.4 30.74 2.906 0.2 32.87 2.723 0.2

u. Hydrogen Fumarate Form III

A2-73 hydrogen fumarate (mono-A2-73 fumarate) Form III is anhydrous,crystalline, melts with a DSC onset temperature of ca. 197° C., and isslightly hygroscopic, picking up ca. 0.9% w/w at 90% RH. Form IIIconsists of large, highly-birefringent columnar (lathe-like) crystals.Form III exhibited moderate solubility at gastric pH (generally about1.5 to 3.5) and lower solubility at GI pH. GI pH shall mean about pH 6in the duodenum, increasing gradually to about pH 7.4 in the terminalileum before dropping to about pH 5.7 in the caecum and graduallyincreasing to about pH 6.7 in the rectum. Measured solubilities of ca.43.0, 4.1 and 12.6 mg/mL at pH 1.2, 4.5 and 6.8, respectively, wereobserved for Form III. The pH of a saturated solution of Form III is3.61. Its PLM and XRPD pattern are provided in FIGS. 31 and 32.

FIG. 31 shows a polarized-light microscopy (PLM) of Anavex2-73 hydrogenfumarate Form III.

XRPD pattern of Anavex2-73 hydrogen fumarate Form III obtained usingcopper Kα radiation is shown in FIG. 32.

The twenty most intense XRPD peaks for Anavex2-73 Fumarate Form III,measured using copper Kα radiation.

TABLE Pos. [° 2θ] d-spacing [Å] Rel. Int. [%] 10.69 8.273 47 11.93 7.417100 12.05 7.342 13 12.14 7.288 20 14.51 6.104 40 14.85 5.967 34 15.785.617 25 15.92 5.566 38 18.97 4.677 67 20.55 4.322 46 20.98 4.235 1821.42 4.145 13 21.49 4.135 18 22.56 3.942 13 23.05 3.859 13 23.25 3.82620 23.69 3.755 23 23.83 3.734 46 23.98 3.711 23 24.82 3.584 14

v. Fumarate Form IV

A2-73 fumarate Form IV is anhydrous, crystalline and melts with a DSConset temperature of ca. 170° C., followed by sublimation. Its XRPDpattern, obtained using copper Kα radiation is provided in FIG. 33.

The twenty most intense XRPD peaks for Anavex2-73 Form fumarate IV,measured using copper Kα radiation.

TABLE Pos. [° 2θ] d-spacing [Å] Rel. Int. [%] 10.72 8.257 43 11.87 7.45673 12.13 7.296 30 13.74 6.444 37 14.52 6.102 13 14.83 5.975 15 15.845.594 32 18.37 4.829 6 21.50 4.133 30 22.84 3.893 71 23.43 3.797 1023.85 3.732 25 24.73 3.600 17 25.81 3.451 12 26.82 3.324 7 27.75 3.21522 28.81 3.099 100 29.44 3.034 38 32.37 2.766 8 34.11 2.628 6

w. Fumarate Form V

A2-73 fumarate (di-A2-73 fumarate) Form V is crystalline. Its XRPDpattern, obtained using copper Kα radiation is provided in FIG. 34.

The twenty most intense XRPD peaks for Anavex2-73 Form fumarate V,measured using copper Kα radiation.

TABLE Pos. [° 2θ] d-spacing [Å] Rel. Int. [%] 9.96 8.884 81 10.71 8.25733 11.92 7.425 100 13.74 6.445 23 14.85 5.967 37 15.83 5.597 44 15.965.555 29 16.69 5.313 42 19.01 4.668 71 19.33 4.591 36 19.99 4.442 2020.60 4.311 21 21.03 4.225 20 21.52 4.129 40 21.68 4.099 19 22.40 3.97035 22.89 3.886 18 23.07 3.855 22 23.29 3.820 30 23.92 3.720 44

II. Pharmaceutical Formulations

One aspect of the disclosure encompasses a pharmaceutical formulationfor delivery of A2-73. A pharmaceutical formulation comprises atherapeutically effective amount of a crystalline form of A2-73 selectedfrom A2-73 freebase and any pharmaceutically acceptable A2-73 salt asdisclosed herein.

A pharmaceutical formulation can be prepared as known in the art forextended or slow release, or for substantially immediate release, andcan comprise from about 1 mg to about 50 g of crystalline A2-73. Forinstance, formulations for immediate delivery can comprise crystallineA2-73 salt such as the hydrochloride salt. In other aspects, theformulations can be prepared for extended release of crystalline A2-73.In non-limiting example, a formulation for extended release ofcrystalline A2-73 can comprise crystalline A2-73 freebase.

A pharmaceutical formulation further comprises one or morepharmaceutically acceptable excipients. Non-limiting examples ofexcipients include chemical enhancers, humectants, pressure sensitiveadhesives, antioxidants, solubilizers, thickening agents, plasticizers,adjuvants, carriers, excipients, vehicles, coatings, and anycombinations thereof. One or more excipients can be selected for oral,transdermal, parenteral, intraperitoneal, intravascular, subcutaneous,by inhalation spray, rectal, or intrapulmonary administration.

Crystalline A2-73 can in general be formulated for improving patientcompliance, preventing a subject from removing the drug-delivery device.For instance, formulations could be formulated for improved patientcompliance and preventing removal of a drug-delivery device by providingformulations for extended delivery. Extended delivery can range forperiods ranging from more than one day, to months. This may beespecially relevant for patients with compromised cognitive and/ormotor-control abilities. Extended delivery for periods can range fromabout 1 day to about 1 year, from about 1 day to about 1 week, fromabout 3 days to about 1 month, from about 2 weeks to about 6 months, orfrom about 2 months to about 4 months.

Extended release formulations could be used for substantially continuousdelivery of drug at a preselected rate. For example, for crystallineA2-73, the drug can be delivered at a rate of from about 1 mg to about100 mg/day, from about 40 to about 60 gm/day, or from about 10 to about30 gm/day. Appropriate amounts of crystalline A2-73 can be readilydetermined by the ordinarily skilled artisan based upon, for example,the intended duration of administration of the drug by the extendedrelease formulation, the delivery mechanism, the particular formulation,and the relative potency of the drug among other factors.

i. Binders

Non-limiting examples of binders suitable for the formulations ofvarious aspects include starches, pregelatinized starches, gelatin,polyvinylpyrrolidone, cellulose, methylcellulose, sodiumcarboxymethylcellulose, ethylcellulose, polyacrylamides,polyvinyloxoazolidone, polyvinylalcohols, C₁₂-C₁₈ fatty acid alcohols,polyethylene glycol, polyols, saccharides, oligosaccharides,polypeptides, oligopeptides, and combinations thereof. The polypeptidemay be any arrangement of amino acids ranging from about 100 to about300,000 Daltons.

The binder can be introduced into the mixture to be granulated in asolid form including but not limited to a crystal, a particle, a powder,or any other finely divided solid form known in the art. Alternatively,the binder can be dissolved or suspended in a solvent and sprayed ontothe mixture in a granulation device as a binder fluid duringgranulation.

ii. Diluent

Non-limiting examples of diluents (also referred to as “fillers” or“thinners”) include carbohydrates, inorganic compounds, andbiocompatible polymers, such as polyvinylpyrrolidone (PVP). Othernon-limiting examples of diluents include dibasic calcium sulfate,tribasic calcium sulfate, starch, calcium carbonate, magnesiumcarbonate, microcrystalline cellulose, dibasic calcium phosphate,tribasic calcium phosphate, magnesium carbonate, magnesium oxide,calcium silicate, talc, modified starches, saccharides such as sucrose,dextrose, lactose, microcrystalline cellulose, fructose, xylitol, andsorbitol, polyhydric alcohols; starches; pre-manufactured directcompression diluents; and mixtures of any of the foregoing.

iii. Disintegrents

Disintegrents can be effervescent or non-effervescent. Non-limitingexamples of non-effervescent disintegrants include starches such as cornstarch, potato starch, pregelatinized and modified starches thereof,sweeteners, clays, such as bentonite, micro-crystalline cellulose,alginates, sodium starch glycolate, gums such as agar, guar, locustbean, karaya, pecitin, and tragacanth. Suitable effervescentdisintegrants include but are not limited to sodium bicarbonate incombination with citric acid, and sodium bicarbonate in combination withtartaric acid.

iv. Preservatives

Non-limiting examples of preservatives include, but are not limited to,ascorbic acid and its salts, ascorbyl palmitate, ascorbyl stearate,anoxomer, N-acetylcysteine, benzyl isothiocyanate, m-aminobenzoic acid,o-aminobenzoic acid, p-aminobenzoic acid (PABA), butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), caffeic acid,canthaxantin, alpha-carotene, beta-carotene, beta-caraotene,beta-apo-carotenoic acid, carnosol, carvacrol, catechins, cetyl gallate,chlorogenic acid, citric acid and its salts, clove extract, coffee beanextract, p-coumaric acid, 3,4-dihydroxybenzoic acid,N,N′-diphenyl-p-phenylenediamine (DPPD), dilauryl thiodipropionate,distearyl thiodipropionate, 2,6-di-tert-butylphenol, dodecyl gallate,edetic acid, ellagic acid, erythorbic acid, sodium erythorbate,esculetin, esculin, 6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline, ethylgallate, ethyl maltol, ethylenediaminetetraacetic acid (EDTA),eucalyptus extract, eugenol, ferulic acid, flavonoids (e.g., catechin,epicatechin, epicatechin gallate, epigallocatechin (EGC),epigallocatechin gallate (EGCG), polyphenol epigallocatechin-3-gallate),flavones (e.g., apigenin, chrysin, luteolin), flavonols (e.g.,datiscetin, myricetin, daemfero), flavanones, fraxetin, fumaric acid,gallic acid, gentian extract, gluconic acid, glycine, gum guaiacum,hesperetin, alpha-hydroxybenzyl phosphinic acid, hydroxycinammic acid,hydroxyglutaric acid, hydroquinone, N-hydroxysuccinic acid,hydroxytryrosol, hydroxyurea, rice bran extract, lactic acid and itssalts, lecithin, lecithin citrate; R-alpha-lipoic acid, lutein,lycopene, malic acid, maltol, 5-methoxy tryptamine, methyl gallate,monoglyceride citrate; monoisopropyl citrate; morin,beta-naphthoflavone, nordihydroguaiaretic acid (NDGA), octyl gallate,oxalic acid, palmityl citrate, phenothiazine, phosphatidylcholine,phosphoric acid, phosphates, phytic acid, phytylubichromel, pimentoextract, propyl gallate, polyphosphates, quercetin, trans-resveratrol,rosemary extract, rosmarinic acid, sage extract, sesamol, silymarin,sinapic acid, succinic acid, stearyl citrate, syringic acid, tartaricacid, thymol, tocopherols (i.e., alpha-, beta-, gamma- anddelta-tocopherol), tocotrienols (i.e., alpha-, beta-, gamma- anddelta-tocotrienols), tyrosol, vanilic acid,2,6-di-tert-butyl-4-hydroxymethylphenol (i.e., Ionox 100),2,4-(tris-3′,5′-bi-tert-butyl-4′-hydroxybenzyl)-mesitylene (i.e., Ionox330), 2,4,5-trihydroxybutyrophenone, ubiquinone, tertiary butylhydroquinone (TBHQ), thiodipropionic acid, trihydroxy butyrophenone,tryptamine, tyramine, uric acid, vitamin K and derivates, vitamin Q10,wheat germ oil, zeaxanthin, or combinations thereof.

v. Flavor-Modifying Agents

Suitable flavor-modifying agents include flavorants, taste-maskingagents, sweeteners, and the like. Flavorants include, but are notlimited to, synthetic flavor oils and flavoring aromatics and/or naturaloils, extracts from plants, leaves, flowers, fruits, and combinationsthereof. Other non-limiting examples of flavors include cinnamon oils,oil of wintergreen, peppermint oils, clover oil, hay oil, anise oil,eucalyptus, vanilla, citrus oils such as lemon oil, orange oil, grapeand grapefruit oil, fruit essences including apple, peach, pear,strawberry, raspberry, cherry, plum, pineapple, and apricot.

Taste-masking agents include but are not limited to cellulosehydroxypropyl ethers (HPC) such as Klucel®, Nisswo HPC and PrimaFloHP22; low-substituted hydroxypropyl ethers (L-HPC); cellulosehydroxypropyl methyl ethers (HPMC) such as Seppifilm-LC, Pharmacoat®,Metolose SR, Opadry YS, PrimaFlo, MP3295A, Benecel MP824, and BenecelMP843; methylcellulose polymers such as Methocel® and Metolose®,Ethylcelluloses (EC) and mixtures thereof such as E461, Ethocel®,Aqualon®-EC, Surelease; Polyvinyl alcohol (PVA) such as Opadry AMB;hydroxyethylcelluloses such as Natrosol®; carboxymethylcelluloses andsalts of carboxymethylcelluloses (CMC) such as Aualon®-CMC; polyvinylalcohol and polyethylene glycol co-polymers such as Kollicoat IR®;monoglycerides (Myverol), triglycerides (KLX), polyethylene glycols,modified food starch, acrylic polymers and mixtures of acrylic polymerswith cellulose ethers such as Eudragit® EPO, Eudragit® RD100, andEudragit® E100; cellulose acetate phthalate; sepifilms such as mixturesof HPMC and stearic acid, cyclodextrins, and mixtures of thesematerials. In other aspects, additional taste-masking agentscontemplated are those described in U.S. Pat. Nos. 4,851,226, 5,075,114,and 5,876,759, each of which is hereby incorporated by reference in itsentirety.

Non-limiting examples of sweeteners include glucose (corn syrup),dextrose, invert sugar, fructose, and mixtures thereof (when not used asa carrier); saccharin and its various salts such as the sodium salt;dipeptide sweeteners such as aspartame; dihydrochalcone compounds,glycyrrhizin; Stevia rebaudiana (Stevioside); chloro derivatives ofsucrose such as sucralose; sugar alcohols such as sorbitol, mannitol,sylitol, hydrogenated starch hydrolysates and the synthetic sweetener3,6-dihydro-6-methyl-1,2,3-oxathiazin-4-one-2,2-dioxide, particularlythe potassium salt (acesulfame-K), and sodium and calcium salts thereof.

vi. Lubricants and Glidants

The lubricant compositions may be utilized to lubricate ingredients thatform a pharmaceutical composition. As a glidant, the lubricantfacilitates removal of solid dosage forms during the manufacturingprocess. Non-limiting examples of lubricants and glidants includemagnesium stearate, calcium stearate, zinc stearate, hydrogenatedvegetable oils, sterotex, polyoxyethylene monostearate, talc,polyethylene glycol, sodium benzoate, sodium lauryl sulfate, magnesiumlauryl sulfate, and light mineral oil. The pharmaceutical compositionwill generally comprise from about 0.01% to about 10% by weight of alubricant. In some aspects, the pharmaceutical composition will comprisefrom about 0.1% to about 5% by weight of a lubricant. In a furtheraspect, the pharmaceutical composition will comprise from about 0.5% toabout 2% by weight of a lubricant.

vii. Dispersants

Dispersants may include but are not limited to starch, alginic acid,polyvinylpyrrolidones, guar gum, kaolin, bentonite, purified woodcellulose, sodium starch glycolate, isoamorphous silicate, andmicrocrystalline cellulose as high hydrophilic-lipophilic balance (HLB)emulsifier surfactants.

viii. Colorants

Depending upon the aspect of the disclosure, it may be desirable toinclude a coloring agent. Suitable color additives include but are notlimited to food, drug and cosmetic colors (FD&C), drug and cosmeticcolors (D&C), or external drug and cosmetic colors (Ext. D&C). Thesecolors or dyes, along with their corresponding lakes, and certainnatural and derived colorants may be suitable for use in various aspectsof the disclosure.

ix. pH Modifiers

Non-limiting examples of pH modifiers include citric acid, acetic acid,tartaric acid, malic acid, fumaric acid, lactic acid, phosphoric acid,sorbic acid, benzoic acid, sodium carbonate and sodium bicarbonate.

(x) Chelating Agents

A chelating agent may be included as an excipient to immobilizeoxidative groups, including but not limited to metal ions, in order toinhibit the oxidative degradation of the morphinan by these oxidativegroups. Non-limiting examples of chelating agents include lysine,methionine, glycine, gluconate, polysaccharides, glutamate, aspartate,and disodium ethylenediaminetetraacetate (Na₂EDTA).

(xi) Antimicrobial Agents

An antimicrobial agent may be included as an excipient to minimize thedegradation of the compound according to this disclosure by microbialagents, including but not limited to bacteria and fungi. Non-limitingexamples of antimicrobials include parabens, chlorobutanol, phenol,calcium propionate, sodium nitrate, sodium nitrite, Na₂EDTA, andsulfites including but not limited to sulfur dioxide, sodium bisulfite,and potassium hydrogen sulfite.

(xii) Release-Controlling Polymers

Release-controlling polymers may be included in the various aspects ofthe solid dosage pharmaceutical compositions incorporating compoundsaccording to this disclosure. In one aspect, the release-controllingpolymers may be used as a tablet coating. In other aspects, includingbut not limited to bilayer tablets, a release-controlling polymer may bemixed with the granules and other excipients prior to the formation of atablet by a known process including but not limited to compression in atablet mold. Suitable release-controlling polymers include but are notlimited to hydrophilic polymers and hydrophobic polymers.

Suitable hydrophilic release-controlling polymers include, but are notlimited to, cellulose acetate, cellulose diacetate, cellulosetriacetate, cellulose ethers, hydroxyethyl cellulose, hydroxypropylcellulose, hydroxypropyl methylcellulose, microcrystalline cellulose,nitrocellulose, crosslinked starch, agar, casein, chitin, collagen,gelatin, maltose, mannitol, maltodextrin, pectin, pullulan, sorbitol,xylitol, polysaccharides, ammonia alginate, sodium alginate, calciumalginate, potassium alginate, propylene glycol alginate, alginate sodiumcarmellose, calcium carmellose, carrageenan, fucoidan, furcellaran,arabic gum, carrageens gum, ghafti gum, guar gum, karaya gum, locustbean gum, okra gum, tragacanth gum, scleroglucan gum, xanthan gum,hypnea, laminaran, acrylic polymers, acrylate polymers, carboxyvinylpolymers, copolymers of maleic anhydride and styrene, copolymers ofmaleic anhydride and ethylene, copolymers of maleic anhydride propyleneor copolymers of maleic anhydride isobutylene), crosslinked polyvinylalcohol and poly N-vinyl-2-pyrrolidone, diesters of polyglucan,polyacrylamides, polyacrylic acid, polyamides, polyethylene glycols,polyethylene oxides, poly(hydroxyalkyl methacrylate), polyvinyl acetate,polyvinyl alcohol, polyvinyl chloride, polystyrenes,polyvinylpyrrolidone, anionic and cationic hydrogels, and combinationsthereof.

(xiii) Coatings

A solid dosage comprising a compound according to this disclosure maycomprise a coating, wherein such a coating may control release of thecompound, act as a moisture barrier, or buffer or modify pH. A “controlreleasing coat” or “controlled release coat” as used herein is definedto mean a functional coat which can for example comprise at least one pHindependent polymer, pH dependent polymer (for example enteric orreverse enteric type polymers), soluble polymer, insoluble polymer,lipids, lipidic materials, or combinations thereof. The coating, whenapplied onto a dosage form, may slow (for example when applied to anormal release matrix dosage form), further slow (for example whenapplied to a controlled release matrix dosage form) or modify the rateof release of a compound according to this disclosure when applied to anuncoated dosage form. For example, the control releasing coat can bedesigned such that when the control releasing coat is applied to adosage form, the dosage form in conjunction with the control releasingcoat can exhibit the release of the compound according to thisdisclosure, such as a “modified-release”, “controlled-release”,“sustained-release”, “extended-release”, “delayed-release”,“prolonged-release” or combinations thereof. The “control releasingcoat” may optionally comprise additional materials that may alter thefunctionality of the control releasing coat.

The term “moisture barrier” as used herein is one, which impedes orretards the absorption of moisture. Compounds according to thisdisclosure may be hygroscopic and, as such, may be susceptible todecomposition over time under highly humid conditions. The proportion ofthe components of the moisture barrier and the amount of the moisturebarrier optionally applied onto the control-releasing coating or ontothe core is typically such that the moisture barrier does not fallwithin the USP definition and requirement for an enteric coat. Suitably,the moisture barrier may comprise an enteric and/or acrylic polymer,suitably an acrylic polymer, optionally a plasticizer, and a permeationenhancer. The permeation enhancer is a hydrophilic substance, whichallows water to enter without physical disruption of the coating. Themoisture barrier may additionally comprise other conventional inertexcipients, which may improve processing of an extended-releaseformulation.

Coating and matrix materials which may be used in accordance with theinvention are those known in the art for use in controlled-releaseformulations, such as synthetic polymers of the polyvinyl type, e.g.polyvinylchloride, polyvinylacetate and copolymers thereof,polyvinylalcohol, and polyvinylpyrrolidone; synthetic polymers of thepolyethylene type, e.g. polyethylene and polystyrene; acrylic acidpolymers; biopolymers or modified biopolymers, such as cellulosicpolymers, shellac and gelatin; fats, oils, higher fatty acids and higheralcohols (i.e., acids and alcohols containing alkyl chains of at least10 carbon atoms), for example aluminum monostearate, cetylalcohol,hydrogenated beef tallow, hydrogenated castor oil, 12-hydroxystearylalcohol, glyceryl mono- or dipalmitate; glyceryl mono-, di- ortristearate; myristyl alcohol, stearic acid, stearyl alcohol, andpolyethyleneglycols; waxes; sugars and sugar alcohols.

The pH-buffering properties of a coating may be strengthened byintroducing into the coating substances chosen from a group of compoundsusually used in antacid formulations, for example magnesium oxide,hydroxide or carbonate, aluminum or calcium hydroxide, carbonate orsilicate; composite aluminum/magnesium compounds, for exampleAl₂O₃.6MgO.CO₂.12H₂O, (Mg₆Al₂(OH)₁₆CO₃.4H₂O), MgO.Al₂O₃.2SiO₂.nH₂O,aluminum bicarbonate coprecipitate or similar compounds; or otherpharmaceutically acceptable pH-buffering compounds, for example thesodium, potassium, calcium, magnesium and aluminum salts of phosphoric,carbonic, citric or other suitable, weak, inorganic or organic acids; orsuitable organic bases, including basic amino acids; and salts orcombinations thereof.

A pH-dependent coating serves to release the drug in desired areas ofthe gastrointestinal (GI) tract, e.g., the stomach or small intestine.When a pH-independent coating is desired, the coating is designed toachieve optimal release regardless of pH-changes in the environmentalfluid, e.g., the GI tract. When the coating is formulated to release acompound according to this disclosure in the intestines (especially theupper small intestines), the coating is often called an “entericcoating”. A pH-dependent coating may include, but is not limited to,acrylic acid polymers and copolymers, for example polymers formed fromacrylic acid, methacrylic acid, methyl acrylate, ammonio methylacrylate,ethyl acrylate, methyl methacrylate and/or ethyl methacrylate (e.g.,Eudragit™); cellulosic polymers such as hydroxypropyl cellulose,hydroxyethyl cellulose, hydroxypropyl methyl cellulose, methylcellulose, ethyl cellulose, cellulose acetate, cellulose acetatephthalate (CAP), cellulose acetate trimellitate, hydroxypropylmethylcellulose phthalate, hydroxypropylmethyl cellulose succinate andcarboxymethylcellulose sodium; shellac (purified lac); vinyl polymersand copolymers such as polyvinyl pyrrolidone, polyvinyl acetate,polyvinylacetate phthalate (PVAP), vinylacetate crotonic acid copolymer,and ethylene-vinyl acetate copolymers; zein; and salts and combinationsthereof.

A. Transdermal Administration

One aspect of the disclosure encompasses formulations of A2-73 fortransdermal administration. Non-limiting examples of transdermalformulations include those used in a transdermal patch, such as but notlimited to gels, ointments, emulsions, microemulsions, aqueous gels,foams, sprays, lotions or creams.

In one aspect, a transdermal formulation of crystalline forms of A2-73is a transdermal patch. A transdermal patch comprises a therapeuticallyeffective amount of a crystalline form of A2-73. The crystalline form ofA2-73 can be A2-73 freebase or A2-73 salt.

The crystalline form of A2-73 in the patch can be A2-73 freebase. Whenthe crystalline form of A2-73 is A2-73 freebase, the patch could containfrom about 40 mg to about 60 mg, from about 80 mg to about 120 mg, orabout 180 mg to about 220 mg of A2-73 freebase. Alternatively, thecrystalline form of A2-73 in the patch can be A2-73 fumarate salt. Whenthe Crystalline form of A2-73 is A2-73 fumarate salt, the patch couldcomprise from about 1 mg to about 55 mg of A2-73 fumarate salt.

Transdermal patches include those formulated for extended or slowrelease, and those formulated for substantially immediate release. Forexample, an extended release transdermal patch may include a crystallineform of A2-73 as a free base or as a A2-73 fumarate salt as disclosedherein. For example, an immediate release patch form may include forexample an A2-73 salt, such as the HCl salt.

The transdermal patch can provide for extended release of A2-73 over aperiod ranging from about 1 day to about 7 days. Additionally, thetransdermal patch can have a transcutaneous maximum flux of A2-73ranging from about 250-350 μg/cm²/h.

The transdermal patch can be a matrix patch or a reservoir patch. In oneaspect, the patch is a matrix patch. Transdermal patches containingamounts of a compound for delivery in a matrix or a reservoir, forextended delivery of the compound are known in the art and can be asdescribed for example in U.S. Pat. No. 9,656,441 and U.S. PatentPublication No. 2019/0099383, the disclosures of which are incorporatedherein in their entirety.

A matrix patch can be covered by a peripheral pressure sensitiveadhesive that extends beyond the patch in all directions. The patch canfurther contain one or more other excipients such as those described inSection III herein and can be selected from chemical enhancers,humectants, pressure sensitive adhesives, antioxidants, solubilizers,thickening agents, plasticizers, and any combinations thereof.

A matrix layer of a transdermal patch can be formulated for extendedrelease. For instance, in addition to comprising a therapeuticallyeffective amount of the active ingredient, a matrix formulation canfurther comprise one or more pharmaceutically acceptable carriers orexcipients. Non-limiting examples of pharmaceutically acceptablecarriers or excipients include chemical penetration enhancers (CPE),chemical enhancers, humectants, pressure sensitive adhesives,antioxidants, solubilizers, thickening agents, plasticizers, and anycombinations thereof.

In some aspects, a matrix layer comprises one or more CPE's.Non-limiting examples of a CPE include anionic surfactants, cationicsurfactants, zwitterionic surfactants, nonionic surfactants, fattyacids, fatty esters, azone and azone-like compounds, ethanol,glycerolmonolaurate, DMF, polyethylenglycole monolaurate, DMSO, ethylalcohol, oleic acid, oleyl alcohol, glycerol monooleate, levulinic acid,dipropylene glycol, diethylene glycol monoethyl ether, lauric lactate,and combinations thereof.

In some aspects a transdermal patch of the disclosure comprises a matrixlayer having a top side and a bottom side, the matrix comprising atherapeutically effective amount of a crystalline form of A2-73 selectedfrom A2-73 freebase and A2-73 salt. The patch also comprises an adhesivelayer having a top side and a bottom side, wherein the bottom side ofthe adhesive layer contacts the top side of the matrix layer, andwherein the adhesive layer having a first portion covering the top sideof the matrix layer and a second portion extending on the sides of thematrix layer. The bottom side of the matrix contacts the skin of a user.

The transdermal patch can further have a protective layer covering thebottom side of the matrix and the bottom side of the second portion ofthe adhesive layer. Further, the transdermal patch can include a coverlayer on the top side of the matrix. Preferably, the cover layer is atleast partially bi-elastic. For instance, the bi-elastic cover layer canan acrylic copolymer having hydroxyl functional groups. In someinstances, the transdermal patch can contain separating layer situatedbetween the top side of the matrix layer and the bottom side of theadhesive layer.

A surface area of the matrix in contact with the skin of a subject canrange from about 1 cm² to about 20 cm², from about 3 cm² to about 5 cm²,or from about 8 cm² to about 10 cm².

In one aspect, a patch comprises a matrix comprising either A2-73freebase or A2-73 fumarate, or a reservoir containing A2-73 freebase orA2-73 fumarate. Other excipients/chemicals/reagents which may beincluded in a patch matrix or reservoir are ethyl oleate (EO) and Tween60, Tween 40, Tween 80, triethanolamine and ethanol, propylene glycol(PG) and polyvinyl alcohol (PVA), polyethylene glycol 400 (PEG 400), andmethanol or any combination thereof. Patch components may includebacking membrane (3M-9720), rate-controlling membrane (3M-CoTran 9728 (2mil) and 9716 (4 mil)), and release liner (SCOTCHPAK 9755), acrylateadhesive Duro-Tak 387/2510.

B. Oral Formulation

Some aspects of the disclosure encompass an oral formulation fordelivery of crystalline forms of A2-73 freebase and salt. Oralformulations are known in the art and include without limitation, atablet, including a suspension tablet, a chewable tablet, aneffervescent tablet or caplet; a pill; a powder such as a sterilepackaged powder, a dispensable powder, and an effervescent powder; acapsule including both soft or hard gelatin capsules such as HPMCcapsules; a lozenge; a sachet; a sprinkle; a reconstitutable powder orshake; a troche; pellets; granules; liquids; suspensions; emulsions; orsemisolids and gels. Alternatively, the pharmaceutical compositions maybe incorporated into a food product or powder for mixing with a liquid,or administered orally after only mixing with a non-foodstuff liquid.

Oral dosage forms include those formulated for extended or slow release,and those formulated for substantially immediate release. For example,an extended release oral dosage form may include a crystalline form ofA2-73 as a free base or as a A2-73 fumarate salt as disclosed herein.For example, an immediate release oral dosage form may include forexample an A2-73 salt, such as the HCl salt. Release characteristics andrelease time can be measured according to methods known in the art.

In one aspect, an oral dosage form comprising crystalline A2-73 providesfor extended release of the A2-73 over a period ranging from about 1 dayto about 3 days, 4-24 hours, e.g., 6-24 hours, preferably 12-24 hours,and can provide for delivery of about 15 to about 30 mg/day of A2-73 toa subject.

In another aspect, an oral dosage form comprising crystalline A2-73provides for substantially immediate release of the A2-73 as understoodin the art, and may for example comprise A2-73 hydrochloride in animmediate-release oral dosage form.

In one aspect, the oral formulation is an enteric coated oral dosageform comprising a core matrix (“core”) comprising a therapeuticallyeffective amount of a crystalline form of A2-73. The crystalline A2-73can be A2-73 freebase or A2-73 salt. The core is surrounded by acoating. Preferably, the coating is an enteric coating

A solid core of the instant disclosure, such as a capsule or tabletformulations contain the crystalline A2-73, along with an excipient.Non-limiting examples of excipients can be as described in Section IIIabove and can include binders, diluents (fillers), disintegrants,effervescent disintegration agents, preservatives (antioxidants),flavor-modifying agents, lubricants and glidants, dispersants, coloringagents, pH modifiers, chelating agents, antimicrobial agents,release-controlling polymers, and combinations of any of these agents.

Non-limiting examples of binders suitable for oral formulations includestarches, pregelatinized starches, gelatin, polyvinylpyrrolidone,cellulose, methylcellulose, sodium carboxymethylcellulose,ethylcellulose, polyacrylamides, polyvinyloxoazolidone,polyvinylalcohols, C₁₂-C₁₈ fatty acid alcohols, polyethylene glycol,polyols, saccharides, oligosaccharides, polypeptides, oligopeptides, andcombinations thereof. The polypeptide may be any arrangement of aminoacids ranging from about 100 to about 300,000 Daltons.

Non-limiting examples of diluents (also referred to as “fillers” or“thinners”) include carbohydrates, inorganic compounds, andbiocompatible polymers, such as polyvinylpyrrolidone (PVP). Othernon-limiting examples of diluents include dibasic calcium sulfate,tribasic calcium sulfate, starch, calcium carbonate, magnesiumcarbonate, microcrystalline cellulose, dibasic calcium phosphate,tribasic calcium phosphate, magnesium carbonate, magnesium oxide,calcium silicate, talc, modified starches, saccharides such as sucrose,dextrose, lactose, microcrystalline cellulose, fructose, xylitol, andsorbitol, polyhydric alcohols; starches; pre-manufactured directcompression diluents; and mixtures of any of the foregoing.

Disintegrents may be effervescent or non-effervescent. Non-limitingexamples of non-effervescent disintegrants include starches such as cornstarch, potato starch, pregelatinized and modified starches thereof,sweeteners, clays, such as bentonite, micro-crystalline cellulose,alginates, sodium starch glycolate, gums such as agar, guar, locustbean, karaya, pecitin, and tragacanth. Suitable effervescentdisintegrants include but are not limited to sodium bicarbonate incombination with citric acid, and sodium bicarbonate in combination withtartaric acid.

Dispersants may include but are not limited to starch, alginic acid,polyvinylpyrrolidones, guar gum, kaolin, bentonite, purified woodcellulose, sodium starch glycolate, isoamorphous silicate, andmicrocrystalline cellulose as high hydrophilic-lipophilic balance (HLB)emulsifier surfactants.

Non-limiting examples of pH modifiers include citric acid, acetic acid,tartaric acid, malic acid, fumaric acid, lactic acid, phosphoric acid,sorbic acid, benzoic acid, sodium carbonate and sodium bicarbonate.

Release-controlling polymers may be included in the oral formulationsincorporating compounds according to this disclosure. In one aspect, therelease-controlling polymers can be used as a tablet coating. In otheraspects, including but not limited to bilayer tablets, arelease-controlling polymer may be mixed with the granules and otherexcipients prior to the formation of a tablet by a known processincluding but not limited to compression in a tablet mold. Suitablerelease-controlling polymers include but are not limited to hydrophilicpolymers and hydrophobic polymers.

A coating may control release of the compound, act as a moisturebarrier, or buffer or modify pH. A “control releasing coat” or“controlled release coat” as used herein is defined to mean a functionalcoat which can for example comprise at least one pH independent polymer,pH dependent polymer (for example enteric or reverse enteric typepolymers), soluble polymer, insoluble polymer, lipids, lipidicmaterials, or combinations thereof. The coating, when applied onto asolid dosage form, may slow (for example when applied to a normalrelease matrix dosage form), further slow (for example when applied to acontrolled release matrix dosage form) or modify the rate of release ofa compound according to this disclosure when applied to an uncoateddosage form. For example, the control releasing coat can be designedsuch that when the control releasing coat is applied to a dosage form,the dosage form in conjunction with the control releasing coat canexhibit the release of the compound according to this disclosure, suchas an “immediate release”, a “modified-release”, “controlled-release”,“sustained-release”, “extended-release”, “delayed-release”,“prolonged-release” or combinations thereof. The coat may optionallycomprise additional materials that may alter the functionality of thecontrol releasing coat.

The pH-buffering properties of a coating may be strengthened byintroducing into the coating substances chosen from a group of compoundsusually used in antacid formulations, for example magnesium oxide,hydroxide or carbonate, aluminum or calcium hydroxide, carbonate orsilicate; composite aluminum/magnesium compounds, for exampleAl₂O₃.6MgO.CO₂.12H₂O, (Mg₆Al₂(OH)₁₆CO₃.4H₂O), MgO.Al₂O₃.2SiO₂.nH₂O,aluminum bicarbonate coprecipitate or similar compounds; or otherpharmaceutically acceptable pH-buffering compounds, for example thesodium, potassium, calcium, magnesium and aluminum salts of phosphoric,carbonic, citric or other suitable, weak, inorganic or organic acids; orsuitable organic bases, including basic amino acids; and salts orcombinations thereof.

A pH-dependent coating serves to release the drug in desired areas ofthe gastrointestinal (GI) tract, e.g., the stomach or small intestine.When a pH-independent coating is desired, the coating is designed toachieve optimal release regardless of pH-changes in the environmentalfluid, e.g., the GI tract. When the coating is formulated to release acompound according to this disclosure in the intestines (especially theupper small intestines), the coating is often called an “entericcoating”. A pH-dependent coating may include, but is not limited to,acrylic acid polymers and copolymers, for example polymers formed fromacrylic acid, methacrylic acid, methyl acrylate, ammonio methylacrylate,ethyl acrylate, methyl methacrylate and/or ethyl methacrylate (e.g.,Eudragit™); cellulosic polymers such as hydroxypropyl cellulose,hydroxyethyl cellulose, hydroxypropyl methyl cellulose, methylcellulose, ethyl cellulose, cellulose acetate, cellulose acetatephthalate (CAP), cellulose acetate trimellitate, hydroxypropylmethylcellulose phthalate, hydroxypropylmethyl cellulose succinate andcarboxymethylcellulose sodium; shellac (purified lac); vinyl polymersand copolymers such as polyvinyl pyrrolidone, polyvinyl acetate,polyvinylacetate phthalate (PVAP), vinylacetate crotonic acid copolymer,and ethylene-vinyl acetate copolymers; zein; and salts and combinationsthereof.

Coating and core materials which may be used in accordance with theinvention are those known in the art for use in controlled-releaseformulations, such as synthetic polymers of the polyvinyl type, e.g.polyvinylchloride, polyvinylacetate and copolymers thereof,polyvinylalcohol, and polyvinylpyrrolidone; synthetic polymers of thepolyethylene type, e.g. polyethylene and polystyrene; acrylic acidpolymers; biopolymers or modified biopolymers, such as cellulosicpolymers, shellac and gelatin; fats, oils, higher fatty acids and higheralcohols (i.e., acids and alcohols containing alkyl chains of at least10 carbon atoms), for example aluminum monostearate, cetylalcohol,hydrogenated beef tallow, hydrogenated castor oil, 12-hydroxystearylalcohol, glyceryl mono- or dipalmitate; glyceryl mono-, di- ortristearate; myristyl alcohol, stearic acid, stearyl alcohol, andpolyethyleneglycols; waxes; sugars and sugar alcohols.

The crystalline A2-73 in the core can be A2-73 freebase, and the corecan comprise from about 1 g to about 50 g of crystalline A2-73. The corecan comprise from about 1 mg to about 50 mg A2-73 freebase. The core canalso comprise from about 1 g to about 50 g, from about 10 mg to about 50mg, from about 20 mg to about 30 mg, or from about 15 mg to about 25 mgof A2-73 fumarate salt. The core can comprise about 35% to about 40% byweight A2-73 freebase or A2-73 fumarate. In one aspect, the corecomprises about 35% to about 40% by weight A2-73 freebase or A2-73fumarate, about 55% to about 70% by weight hydroxypropyl methylcelluloseacetate succinate, about 0.3% to about 0.9% by weight magnesiumstearate, and about 0.05% to about 0.5% by weight colloidal silicondioxide. The hydroxypropyl methylcellulose acetate succinate is solublein aqueous solutions with a pH of about 5.5 and greater, a second gradeof hydroxypropyl methylcellulose acetate succinate is soluble in aqueoussolutions with a pH of about 6.8 and greater, and combinations thereof.

C. Subcutaneous

The formulation can be a subcutaneous injectable dosage formulation. Insome aspects, the formulation can be an extended delivery subcutaneousinjectable dosage formulation or for substantially immediate delivery.Extended release subcutaneous dosage formulations can comprising fromcomprise from about 0.1 to about 5 g of crystalline A2-73 to about 0.5 gto about 3 g of crystalline A2-73.

Injectable dosage formulations for extended release of a drug are knownin the art and can include an injectable formulation formulated forextended delivery of a drug such as implantable drug delivery devices.The term “drug delivery device” as used herein refers to any implantabledevice suitable for delivering a formulation according to thedisclosure. Non-limiting examples of devices include any implantabledevice with any mechanism of action including diffusive, erodible, orconvective systems, e.g., osmotic pumps, biodegradable implants, electrodiffusion systems, electro osmosis systems, vapor pressure pumps,electrolytic pumps, effervescent pumps, piezoelectric pumps,erosion-based systems, or electromechanical systems.

III. Dosage Forms

One aspect of the disclosure encompasses dosage forms of A2-73. A dosageform contains a therapeutically effective amount of A2-73 in acrystalline form. For instance, a dosage form can contain aneuroprotective amount of crystalline A2-73. In some aspects, theneuroprotective amount is an anti-neurodegenerative amount of A2-73 in acrystalline form as disclosed herein. The crystalline A2-73 can becrystalline A2-73 freebase or crystalline A2-73 salt. The dosage formscan be formulated as described in Section II above.

A dosage form can comprise from about 1 mg to about 50 g, from about 1mg to about 500 mg, or about 1 mg to about 100 mg of A2-73 freebase orA2-73 salt. Further,

Dosage forms can comprise from about 1 mg to about 500 mg, from about 50to about 400 mg, from about from about 75 to about 150 mg, or from about150 to about 200 mg of A2-73 freebase or a A2-73 salt. For instance, thedosage form can comprise 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100,120, 140, 160, 180, 200, 220, 240, 260, 280, or 300 or more mg of A2-73freebase or A2-73 salt. In some aspects, dosage forms can comprise fromabout 1 mg to about 500 mg, or about 1 mg to about 100 mg of A2-73freebase or A2-73 salt.

In some aspects, the crystalline form of A2-73 in the dosage form is afreebase. When the A2-73 is a freebase, the dosage form can comprisefrom about 1 mg to about 500 mg, from about 40 mg to about 60 mg, fromabout 80 mg to about 120 mg, or about 180 mg to about 220 mg of A2-73freebase.

In other aspects, the crystalline form of A2-73 in the dosage form is asalt of A2-73. When the A2-73 is a salt, the dosage form can comprisefrom about 1 mg to about 500 mg, about 1 mg to about 55 mg, from about40 mg to about 60 mg, from about 80 mg to about 120 mg, or about 180 mgto about 220 mg of A2-73 salt.

Dosage forms include those formulated for extended or slow release, andthose formulated for immediate release. For example, an immediaterelease dosage form may include a crystalline form of A2-73 as the freebase or as a A2-73 salt as disclosed herein. For example, afast-dissolve oral dosage form may include for example an A2-73 salt,such as the HCl salt. Alternatively, a dosage form may include acrystalline form of A2-73 as the free base or as a A2-73 salt formulatedfor inhalation drug delivery, either as a dry powder or aerosol spray.

Dosage forms also include those formulated for topical administration.For instance, a dosage form can be formulated as one or more of a gel,ointment, emulsion, microemulsion, solution, suspension, paste, gel,foam, spray, lotion, or cream. In one aspect, a topical administrationdosage form is a transdermal patch. Transdermal patches can be forexample as described in Section III below. When the dosage form isformulated as a transdermal patch, the transdermal patch can containfrom about 40 mg to about 60 mg, from about 80 mg to about 120 mg, orfrom about 180 mg to about 220 mg of A2-73 freebase in crystalline form.

Dosage forms can alternatively be formulated for oral administration.Dosage forms formulated for oral administration can be tablets toswallow, chew, or dissolve in water or under the tongue, capsules andchewable capsules, powders, granules, teas, drops, or liquid medicationsor syrups. Preferably, the dosage form is an enteric coated oralformulation. Enteric coated oral formulations can be as described inSection IV below.

When the dosage form is an enteric coated oral formulation, theformulation can comprise from about 0.1 mg to about 60 mg A2-73freebase, preferably from about 1 mg to about 50 mg A2-73 freebase.

An enteric coated oral formulation can also contain A2-73 salt incrystalline form. The A2-73 salt can be a fumarate salt, a sulfate salt,a mesylate salt, a dihydrogen phosphate salt, an edisylate salt, abenzoate salt, a hydrochloride salt, and an oxalate salt. In one aspect,the A2-73 salt is a fumarate salt. When the A2-73 salt is a fumaratesalt, the enteric coated oral formulation can comprise from about 0.1 toabout 100 mg of A2-73 fumarate salt, preferably from about 1 mg to about55 mg of A2-73 fumarate salt.

Dosage forms also encompass those formulated for subcutaneous and/orintramuscular injection. For example, an intramuscular dosage form maycomprise A2-73 in the free base form, dissolved in an oil matrix forintramuscular injection, or alternatively prepared as a suspension ofthe free base for intramuscular injection. A dosage form formulated forsubcutaneous or intramuscular injection may comprise A2-73 in a salt orfree base form as disclosed herein, prepared as microspheres usingmethods known in the art. Alternatively, A2-73 in free base or salt formmay be coated, for example using Atomic Layer Deposition (ALD)techniques, with a thin layer coating such as a coating of zinc oxide,and used in a formulation for subcutaneous or intramuscular injection.Alternatively, A2-73 free base may be dissolved in a biodegradablepolymer matrix, and then implanted subcutaneously (or used in atransdermal patch as detailed further below).

IV. Methods of Administering A2-73 and Methods of Treating

One aspect of the disclosure encompasses a method of administering A2-73to a subject. The method comprises administering the A2-73 to thesubject in a dosage form comprising a crystalline form of A2-73 selectedfrom A2-73 freebase and A2-73 salt.

Dosage forms can be as described in Section III above. The dosage formscan be formulated for immediate or extended release, and can beformulated for oral, transdermal, subcutaneous, or other forms ofadministration. Formulations can be as described in Section II above.

Extended release dosage forms comprising crystalline A2-73 canadminister A2-73 over a period of about two weeks, 30 days, about 45days, about 60 days, about 90 days, or about 120 to about 180 days 120to about 180 days.

In some aspects, the method comprises administering a topical dosageform. The topical dosage form can be a transdermal patch. Thetransdermal patch can be replaced daily, weekly, or longer. In someaspects, the transdermal patch can maintain a level of A2-73 in theblood of the subject for a period of time ranging from about 5 ng/ml toabout 15 ng/ml and particularly about 10 ng/ml can be maintained.

The crystalline form of A2-73 can also be administered orally using adosage form formulated for oral administration. Preferably, a dosageform is an enteric coated oral formulation.

The enteric coated oral dosage form can be administered every other day.The dosage form can deliver about 15 to about 30 mg/day of A2-73.Further, when formulated for extended release of A2-73, the oral dosageform could deliver A2-73 for a period of time can range from about 1 dayto about 7 days, about 48 hours, about 72 hours, or greater.

One aspect of the disclosure encompasses a method of treatingAlzheimer's disease in a subject in need thereof, the method comprisingadministering a dosage form comprising a therapeutically effectiveamount of a crystalline form of A2-73 selected from A2-73 freebase andA2-73 salt.

One aspect of the disclosure encompasses a method of treating aprogressive dementia in a subject in need thereof, the method comprisingadministering a dosage form comprising a therapeutically effectiveamount of a crystalline form of A2-73 selected from A2-73 freebase andA2-73 salt.

V. Treating a Neurodegenerative Disease

Sig-1R expression or activity are linked to neurodegeneration, and theactivation of Sig-1R is associated with neuroprotection in different invitro and in vivo models, employing different types of pharmacologicalSig-1R activators with different pharmacological profiles. The inventorshave surprisingly discovered that A2-73, a mixed muscarinic receptorligand and Sig-1R agonist, can be used to treat neurodegenerativedisease. As such, any of the crystalline forms of A2-73, and topical andoral dosage forms as disclosed can be administered to a subject in needthereof, for neuroprotection including treatment of a neurodegenerativedisease.

As such, one aspect of the disclosure encompasses a pharmaceuticalcomposition for the treatment of a neurodegenerative disease. Thecomposition comprises an anti-neurodegenerative effective amount ofA2-73. The A2-73 can be crystalline polymorph of A2-73, and can be afreebase or a salt. Preferably, the A2-73 is a hydrochloride salt ofA2-73.

The anti-neurodegenerative effective amount can range from about 0.5 mgto about 20 mg, about 1 mg to about 60 mg, about 30 mg to about 50 mg,or about 3 mg to about 5 mg.

Another aspect of the disclosure encompasses a dosage form comprising anamount of A2-73 effective for the treatment of a neurodegenerativedisease. The amount of A2-73 in the dosage form can be from about 0.01to about 10 mg/kg.

Another aspect of the disclosure encompasses a method of treating ananti-neurodegenerative disease in a subject in need thereof comprisingadministering to the subject an anti-neurodegenerative effective amountof A2-73. The neurodegenerative disease can be selected from Alzheimer'sdisease, Parkinson's disease, prion diseases, Huntington's disease,motor neuron diseases (MND) such as amyotrophic lateral sclerosis,spinocerebellar ataxia (SCA), and spinal muscular atrophy (SMA).

The anti-neurodegenerative effective amount of A2-73 can range fromabout 0.5 mg/day to about 100 mg/day, from about 1 to about 60 mg/day,from about 20 to about 50 mg/day, from about 20 to about 30 mg/day, orfrom about 15 to about 25 mg/day. Administering theanti-neurodegenerative effective amount of A2-73 can provide bloodlevels of about 10 ng/ml, about 12 ng/ml, about of A2-73.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art towhich this invention belongs. The following references provide one ofskill with a general definition of many of the terms used in thisinvention: Singleton et al., Dictionary of Microbiology and MolecularBiology (2nd ed. 1994); The Cambridge Dictionary of Science andTechnology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R.Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, TheHarper Collins Dictionary of Biology (1991). As used herein, thefollowing terms have the meanings ascribed to them unless specifiedotherwise.

When introducing elements of the present disclosure or the preferredaspects(s) thereof, the articles “a”, “an”, “the” and “said” areintended to mean that there are one or more of the elements. The terms“comprising”, “including” and “having” are intended to be inclusive andmean that there may be additional elements other than the listedelements.

As various changes could be made in the above-described cells andmethods without departing from the scope of the invention, it isintended that all matter contained in the above description and in theexamples given below, shall be interpreted as illustrative and not in alimiting sense.

The term “comprising” means “including, but not necessarily limited to”;it specifically indicates open-ended inclusion or membership in aso-described combination, group, series and the like. The terms“comprising” and “including” as used herein are inclusive and/oropen-ended and do not exclude additional, unrecited elements or methodprocesses. The term “consisting essentially of” is more limiting than“comprising” but not as restrictive as “consisting of.” Specifically,the term “consisting essentially of” limits membership to the specifiedmaterials or steps and those that do not materially affect the essentialcharacteristics of the claimed invention.

The term “subject” as used herein refers to a mammalian subject,including without limitation a human, a non-human primate, a mouse, arat, guinea pig, and a dog.

As used herein, the terms “extended” or “slow” release or delivery areused interchangeably, and can be understood in contrast to an immediaterelease composition. In an extended release formulation, the activeingredient is gradually, continuously liberated over time, at a rateappropriate for the intended use of the dosage form. In particular, theterm indicates that the formulation does not release the full dose ofthe active ingredient immediately after dosing, and that the formulationallows a reduction in dosage frequency. A slow or extended release, usedsynonymously with prolonged action, sustained release, or modifiedrelease, dosage form is a dosage form that allows a reduction in dosingfrequency or a significant increase in patient compliance or therapeuticperformance as compared to that presented as an immediate release dosageform (e.g., as a solution or an immediate drug-releasing, conventionalsolid dosage form).

EXAMPLES

The publications discussed above are provided solely for theirdisclosure before the filing date of the present application. Nothingherein is to be construed as an admission that the invention is notentitled to antedate such disclosure by virtue of prior invention.

The following examples are included to demonstrate the disclosure. Itshould be appreciated by those of skill in the art that the techniquesdisclosed in the following examples represent techniques discovered bythe inventors to function well in the practice of the disclosure. Thoseof skill in the art should, however, in light of the present disclosure,appreciate that many changes could be made in the disclosure and stillobtain a like or similar result without departing from the spirit andscope of the disclosure, therefore all matter set forth is to beinterpreted as illustrative and not in a limiting sense.

Example 1 Preparation of Form I, Hydrochloride Salt

Form I can be obtained via crystallization of Anavex2-73 from anhydroussolvents, for example, isopropyl alcohol (IPA). At 70° C., Form I can beobtained, for example, from IPA containing up to at least 2.5% v/vwater. Form I can also be obtained via sublimation. Some examples ofForm I preparation follow:

(i) Example 1.

Approximately 100 mg of Anavex2-73 was weighed into a sample vial, towhich 0.5 mL or 1 mL of 2-ethoxyethanol, 1-propanol, acetone,acetonitrile, dichloromethane, dimethyl sulfoxide, ethanol,N,N′-dimethylacetamide, dimethylformamide, N-methyl-2-pyrrolidone ortert-butanol was added. To these vials additional Anavex2-73 was added,if needed, to ensure a mobile slurry was observed. The slurries wereagitated for ca. 72 hours using an incubator shaker with temperaturecycling employed between ambient temperature (about 20 to 25° C.) and40° C. (2 hours at each temperature). After ca. 72 hours of temperaturecycling the saturated solutions were separated from the slurries using a0.45 μm syringe filter. To approximately ¼^(th) of each filtrate,tert-butyl methyl ether was added to precipitate out Form I, which wascharacterized by XRPD.

(ii) Example 2.

Approximately 100 mg of Anavex2-73 was weighed into a sample vial, towhich 0.5 mL or 1 mL of 2-ethoxyethanol, 1-propanol, acetone,acetonitrile, dichloromethane, dimethyl sulfoxide, ethanol,N,N′-dimethylacetamide, dimethylformamide, N-methyl-2-pyrrolidone ortert-butanol was added. To these vials additional Anavex2-73 was added,if needed, to ensure a mobile slurry was observed. The slurries wereagitated for ca. 72 hours using an incubator shaker with temperaturecycling employed between ambient temperature and 40° C. (2 hours at eachtemperature). After ca. 72 hours of temperature cycling, the slurrieswere filtered through a 0.45 μm filter to isolate the Form Iprecipitate, which was characterized by XRPD.

(iii) Example 3.

Anavex2-73 is slurried in IPA containing ca. 2.5% v/v water (or less)and stirred for 20 hrs. at 70° C. After which, the Form I precipitate isisolated by filtration, which was characterized by XRPD.

(iv) Example 4.

Approximately 100 mg of Anavex2-73 was heated to approximately 200° C.in a 5 mL beaker, topped with ice, under atmospheric pressure. Theresidue was collected and characterized by XRPD.

Example 2 Preparation of Form II, Hydrochloride Salt

Form II can be obtained via crystallization of Anavex2-73 from organicsolvent:water mixtures, for example, isopropyl alcohol (IPA):water(90:10). It can also be obtained via air evaporation at ambienttemperature from 1-butanol, chloroform, ethanol or tert-butanol.Examples of Form II preparation follow:

(i) Example 1.

Approximately 100 mg of Anavex2-73 was weighed into a sample vial, towhich 0.5 mL or 1 mL of water, IPA:water (95:5 v/v) or IPA:water(97.5:2.5 v/v) was added. To this vial additional Anavex2-73 was added,if needed, to ensure a mobile slurry was observed. The slurries wereagitated for ca. 72 hours using an incubator shaker with temperaturecycling employed between ambient temperature and 40° C. (2 hours at eachtemperature). After ca. 72 hours of temperature cycling the saturatedsolutions were separated from the slurries using a 0.45 μm syringefilter. To approximately ¼^(th) of each filtrate, tert-butyl methylether, or in the case of water, THF was added to precipitate out FormII, which was characterized by XRPD.

(ii) Example 2.

Approximately 100 mg of Anavex2-73 was weighed into a sample vial, towhich 0.5 mL or 1 mL of IPA containing 2.5 to 10% v/v water. To thesevials additional Anavex2-73 was added, if needed, to ensure a mobileslurry was observed. The slurries were agitated for ca. 72 hours usingan incubator shaker with temperature cycling employed between ambienttemperature and 40° C. (2 hours at each temperature). After ca. 72 hoursof temperature cycling, the slurries were filtered through a 0.45 μmfilter to isolate the Form II precipitate, which was characterized byXRPD.

(iii) Example 3.

Anavex2-73 was slurried in IPA containing ca. 2.5 to 92.5% v/v water andstirred for 20 hrs at a temperature of 20° C. After which, the Form IIprecipitate was isolated by filtration, which was characterized by XRPD.

(iv) Example 4.

Anavex2-73 is slurried in IPA containing ca. 7.5 to 45% v/v water andstirred for 20 hrs. at a temperature of 70° C. After which, the Form IIprecipitate is isolated by filtration, which was characterized by XRPD.

(v) Example 5.

Form II was produced on a 250 mg scale using both IPA:water (95:5% v/v)and IPA:water (97.5:2.5% v/v). Approximately 250 mg of Anavex2-73 wasweighed into 20 mL scintillation vials. To each vial was added 2.5 mL ofeither IPA:water (95:5% v/v) or IPA:water (97.5:2.5% v/v). The slurrieswere agitated for ca. 48 hours using an incubator shaker withtemperature cycling employed between ambient temperature and 40° C. (2hours at each temperature). After ca. 48 hours of temperature cyclingthe saturated solutions were separated from the slurries. The saturatedsolutions were then allowed to evaporate at ambient temperature. Theresidual solid material after temperature cycling was analyzed by XRPD.The residual solid was allowed to air dry at ambient temperature, andonce again, analyzed by XRPD.

Example 3 Preparation of Form III, Chloride Salt

Form III can be obtained via crystallization of Anavex2-73 from waterand isopropyl alcohol (IPA) at 20° C.

In some aspects, preparation of Form III follows:

(i) Example 1.

In one aspect, an example of preparation of Form III is: Approximately100 mg of Anavex2-73 was weighed into a sample vial, to which 0.5 mL ofwater was added. To this vial additional Anavex2-73 was added to ensurea mobile slurry was observed. The slurry was agitated for ca. 72 hoursusing an incubator shaker with temperature cycling employed betweenambient temperature and 40° C. (2 hours at each temperature). After ca.72 hours of temperature cycling the saturated solution was separatedfrom the slurry using a 0.45 μm syringe filter. Approximately ¼th of thefiltrate was allowed to evaporate at ambient temperature (ca. 20° C.).XRPD data was collected for solid residue.

(ii) Example 2.

In another aspect, an example of preparation of Form III is: Anavex2-73is slurried in IPA and stirred for 20 hours at a temperature of 20° C.After which, the Form III precipitate is isolated by filtration andcharacterized by XRPD.

(iii) Example 3.

In one aspect, an example of preparation of Form III is: Form IIImaterial was produced on a 500 mg scale using water. Approximately 500mg of Anavex2-73 was weighed into a 20 mL scintillation vial and 600 μLof water added. The slurry was then agitated for ca. 48 hours using anincubator shaker with temperature cycling employed between ambienttemperature and 40° C. (2 hours at each temperature). After ca. 24 hoursa thin slurry was noted and an additional 130 mg of Anavex2-73 was addedto the slurry. After the full 48 hours of temperature cycling thesaturated solution was separated from the slurry. The saturated solutionwas then allowed to evaporate at ambient temperature. The residual solidmaterial after temperature cycling was analyzed by XRPD. The residualsolid was allowed to air dry at ambient temperature and re-analyzed byXRPD.

Example 4 Preparation of Form IV, Hydrochloride Salt

Form IV was obtained via Anavex2-73 lyophilization from water. In oneaspect, preparation of Form IV follows:

(i) Example 1.

Approximately 20 mg of Anavex2-73 was weighed into a 2 mL sample vialand dissolved in 200 μL deionized water. The sample was then placed in afreezer at −20° C. Once frozen, the sample was lyophilized andcharacterized by XRPD to assess the crystallinity of the material.

Example 5 Preparation of Form V, Hydrochloride Salt

Form V was obtained via Anavex2-73 rotary evaporation fromdichloromethane. In one aspect, preparation of Form V follows:

(i) Example 1.

Approximately 20 mg of Anavex2-73 was dissolved in 300 μL ofdichloromethane and allowed to rapidly evaporate in a fume-hood,utilizing a rotary evaporator. XRPD data was collected for the resultingsolid material.

Example 6 Preparation of Form VI, Hydrochloride Salt

Form VI was obtained upon rapid cooling of an aqueous solution ofAnavex2-73 to 5° C. Specific example of Form VI preparation follows:

(i) Example 1.

Approximately 100 mg of Anavex2-73 was weighed into a sample vial, towhich 0.5 mL of water was added. To this vial additional Anavex2-73 wasadded to ensure a mobile slurry was observed. The slurry was agitatedfor ca. 72 hours using an incubator shaker with temperature cyclingemployed between ambient temperature and 40° C. (2 hours at eachtemperature). After ca. 72 hours of temperature cycling the saturatedsolution was separated from the slurries using a 0.45 μm syringe filter.Approximately ¼th of the filtrate was placed in a 5° C. refrigerator andstored until solid precipitate was observed. XRPD data was collected forthe damp precipitate to prevent potential desolvation.

Example 7 Preparation of Form VII, Hydrochloride Salt

Form VII was obtained via air evaporation of Anavex2-73 from methanol.Specific example of Form VII preparation follows:

(i) Example 1.

Approximately 100 mg of Anavex2-73 was weighed into a sample vial, towhich 0.5 mL of methanol was added. To this vial additional Anavex2-73was added to ensure a mobile slurry was observed. The slurry wasagitated for ca. 72 hours using an incubator shaker with temperaturecycling employed between ambient temperature and 40° C. (2 hours at eachtemperature). After ca. 72 hours of temperature cycling the saturatedsolution was separated from the slurry using a 0.45 μm syringe filter.Approximately ¼^(th) of the filtrate was allowed to evaporate at ambienttemperature (ca. 20° C.). XRPD data was collected for solid residue.

Example 8 Preparation of Form VIII, Hydrochloride Salt

Form VIII is obtained by slurrying Anavex2-73 in water at 20° C.Specific examples of Form VIII preparation follow:

(i) Example 1.

Anavex2-73 was added to ca. 1.5 mL of water at 20° C. until a slurry wasobtained, with the suspended material analyzed using XRPD afterslurrying for 20 hrs.

(ii) Example 2.

Anavex2-73 is completely dissolved in water and allowed to air evaporateat 20° C. until precipitation is observed. The resulting Form VIII isisolated while still wet with water, as once dried, it converts to FormI. XRPD patterns were collected pre/post-drying.

Example 9 Preparation of Form I, Freebase

Into a 250 mL separating funnel, 150 mL of EtOAc and 500 mg of A2-73(hydrochloride salt) were added, followed by 100 mL of concentratedNaHCO₃. The resulting mixture was shaken and the aqueous layer wasremoved. Two additional 100 mL aliquots of concentrated NaHCO₃ wereadded, and each time, the mixtures were shaken, and the aqueous layerremoved.

The organic layer was washed with 100 mL of deionized water, and thenthe organic layer was dried using magnesium sulphate, then filtered. Thefiltrate was collected and the EtOAc was removed using a rotaryevaporator. A clear oil was obtained post-evaporation, which was driedunder a stream of nitrogen, yielding a white solid. The resulting solidwas weighed and analyzed by XRPD.

Data show A2-73 freebase is (i) crystalline, (ii) not highly watersoluble (unlike the HCl salt), and (iii) non-hygroscopic. Its molecularweight (MW) of 280 is below the accepted general transdermal cutoff MWof about 400, and its calculated LogP is 3.5. Additionally, as A2-73freebase only has 2 hydrogen bond donor/acceptor sites, it is under thegeneral rule-of-thumb limit of about 5 hydrogen bond donor/acceptorsites, which would potentially restrict transdermal delivery.

Therefore, A2-73 freebase is a useful active pharmaceutical ingredientin a transdermal extended-release formulation. Reference is made to anoral dose of 20 mg of A2-73. Transdermal dosing 2× per week at asignificantly lower administration dose based in, it is believed,avoiding first-pass liver or hepatic first pass effect is a usefulresult of the present invention. Transdermal patch matrix layer mayusefully contain, by way of non-limiting example, oleyl oleate, povidoneK90, levulinic acid, crosslinked poly [acrylicacid-co-butylacrylate-co-(2-ethylhexyl)acrylate-co-vinylacetate]. Alsonoted are absorption enhancers (penetration enhancers) in the matrixformulation to generate a high flux of the active compounds when thesystem is applied to the skin. Typical known enhancers are ethanol,glycerolmonolaurate, DMF, polyethylenglycole monolaurate, etc.

Example 10 Preparation of Sulfate Form I

A2-73 sulfate Form I was obtained via addition of sulfuric acid (in THF)to A2-73 freebase in ethanol, THF, acetone, 2-propanol or2-ethoxyethanol. Specific examples of A2-73 sulfate Form I preparationfollow:

(i) Example 1.

Approximately 200 mg of A2-73 freebase was weighted into a 20 mLscintillation vial, after which, 4 mL of ethanol was added to the vial.The apparent pH of the resulting solution was determined using a pHmeter. Sulfuric acid stock solution (1119.4 μL of a solution containing4.1 μL of 98% sulfuric acid per 74.6 μL of THF) was added to the vial,followed by agitation. The apparent pH of the solution was once againmeasured. The sample was then temperature-cycled between ambient and 40°C. for ca. 24 hours. As no solid was observed at the end of ca. 24hours, the sample was left uncapped in a fume hood and allowed toair-evaporate for ca. 72 hours, followed by drying under a nitrogenstream. As no solid was observed, the sample was placed in a vacuum ovenfor one hour, resulting in a colorless, sticky solid. The sample wasfurther dried in a vacuum oven for ca. 4 hours, and a white solid wasobtained, which was characterized by XRPD.

(ii) Example 2.

A stock solution of sulfuric acid was prepared in THF (272.0 μL ofsulfuric acid in 4728.0 μL THF). Separately, 20 mg of the A2-73 freebasewas weighed into 1.5 mL HPLC vials, and to each vial, 300 μL of theappropriate solvent (THF, ethanol, acetone, 2-propanol or2-ethoxyethanol) was added, along with 74.6 μL of acid stock solution(1.05 equivalents of acid). The samples were temperature-cycled betweenambient and 40° C. in 4-hour cycles for 72 hours. XRPD data wascollected for the isolated precipitate.

Example 11 Preparation of Sulfate Form II

A2-73 sulfate Form II was obtained via addition of sulfuric acid (inTHF) to A2-73 freebase in acetonitrile. Specific example of A2-73sulfate Form II preparation follows:

(i) Example 1.

A stock solution of sulfuric acid was prepared in THF (272.0 μL ofsulfuric acid in 4728.0 μL THF). Separately, 20 mg of the A2-73 freebasewas weighed into a 1.5 mL HPLC vial, and 300 μL of acetonitrile wasadded, along with 74.6 μL of acid stock solution (1.05 equivalents ofacid). The sample was temperature-cycled between ambient and 40° C. in4-hour cycles for 72 hours, and the resulting precipitate was analyzedby XRPD.

Example 12 Preparation of Mesylate Form I

A2-73 mesylate Form I was obtained via addition of methane sulfonic acid(in THF) to A2-73 freebase in ethanol, acetonitrile, acetone, 2-propanolor 2-ethoxyethanol. Specific example of A2-73 mesylate Form Ipreparation follows:

(i) Example 1.

A stock solution of methane sulfonic acid was prepared in THF (324.4 μLof methane sulfonic acid in 4675.6 μL THF). Separately, 20 mg of theA2-73 freebase was weighed into 1.5 mL HPLC vials, to which 300 μL ofthe appropriate solvent (ethanol, acetonitrile, acetone, 2-propanol or2-ethoxyethanol) was added, along with 74.6 μL of acid stock solution(1.05 equivalents of acid). The sample was temperature-cycled betweenambient and 40° C. in 4-hour cycles for 72 hours. The samples werefiltered, and approximately 100 μL of the mother liquor from each saltformation reaction was added to 2 mL glass vials. The vials were leftuncapped in a cupboard to allow evaporation. Observed solidspost-evaporation were analyzed by XRPD.

Example 13 Preparation of Oxalate Form I

A2-73 oxalate Form I was obtained via addition of oxalic acid (in THF)to A2-73 freebase in THF. Specific example of A2-73 oxalate Form Ipreparation follows:

(i) Example 1.

A stock solution of oxalic acid was prepared in THF (450.2 μL of oxalicacid in 4549.8 μL THF). Separately, 20 mg of the A2-73 freebase wasweighed into a 1.5 mL HPLC vial, to which 300 μL of THF was added, alongwith 74.6 μL of acid stock solution (1.05 equivalents of acid). Thesample was temperature-cycled between ambient and 40° C. in 4-hourcycles for 72 hours, and the resulting precipitate was analyzed by XRPD.

Example 14 Preparation of Oxalate Form II

A2-73 oxalate Form II was obtained via addition of oxalic acid (in THF)to A2-73 freebase in acetone. Specific example of A2-73 oxalate Form IIpreparation follows:

(i) Example 1.

A stock solution of oxalic acid was prepared in THF (450.2 μL of oxalicacid in 4549.8 μL THF). Separately, 20 mg of the A2-73 freebase wasweighed into a 1.5 mL HPLC vial, to which 300 μL of acetone was added,along with 74.6 μL of acid stock solution (1.05 equivalents of acid).The sample was temperature-cycled between ambient and 40° C. in 4-hourcycles for 72 hours, and the resulting precipitate was analyzed by XRPD.

Example 15 Preparation of Oxalate Form III

A2-73 oxalate Form III was obtained via addition of oxalic acid (in THF)to A2-73 freebase in ethanol. Specific examples of A2-73 oxalate FormIII preparation follow:

(i) Example 1.

Approximately 200 mg of A2-73 freebase was weighted into a 20 mLscintillation vial, after which, 4 mL of ethanol was added to the vial.The apparent pH of the resulting solution was determined using a pHmeter. Oxalic acid stock solution (1119.4 μL of a solution containing6.72 mg of phosphoric acid per 74.6 μL of THF) was added (1.05equivalents) to the vial, followed by agitation. The apparent pH of thesolution was once again measured. The sample was then temperature-cycledbetween ambient and 40° C. for ca. 24 hours, with precipitated solidisolated at the end of ca. 24 hours and air-dried for ca. 72 hours,followed by characterization by XRPD.

(ii) Example 2.

A stock solution of oxalic acid was prepared in THF (450.2 μL of oxalicacid in 4549.8 μL THF). Separately, 20 mg of the A2-73 freebase wasweighed into a 1.5 mL HPLC vial, to which 300 μL of ethanol was added,along with 74.6 μL of acid stock solution (1.05 equivalents of acid).The sample was temperature-cycled between ambient and 40° C. in 4-hourcycles for 72 hours, and the resulting precipitate was analyzed by XRPD.

(iii) Example 3.

A stock solution of oxalic acid was prepared in THF (450.2 μL of oxalicacid in 4549.8 μL THF). Separately, 20 mg of the A2-73 freebase wasweighed into a 1.5 mL HPLC vial, to which 300 μL of ethanol was added,along with 74.6 μL of acid stock solution (1.05 equivalents of acid).The sample was temperature-cycled between ambient and 40° C. in 4-hourcycles for 72 hours. The sample was filtered and approximately 100 μL ofthe filtrate was added to a 2 mL glass vial. The vial was left uncappedin a cupboard to allow evaporation. Observed solid post-evaporation wasanalyzed by XRPD.

(iv) Example 4.

A stock solution of oxalic acid was prepared in THF (450.2 μL of oxalicacid in 4549.8 μL THF). Separately, 20 mg of the A2-73 freebase wasweighed into a 1.5 mL HPLC vial, to which 300 μL of ethanol was added,along with 74.6 μL of acid stock solution (1.05 equivalents of acid).The sample was temperature-cycled between ambient and 40° C. in 4-hourcycles for 72 hours. The sample was filtered and approximately 100 μL offiltrate was measured into a 1.5 mL HPLC vial. The vial was capped andplaced in a fridge at ca. 5° C. for ca. 24 hours. The sample was checkedperiodically and observed solid was analyzed by XRPD. If the sampleremained a solution it was placed in a freezer at ca. −20° C. for ca. 24hours. The sample was checked periodically and observed solid wasanalyzed by XRPD.

(v) Example 5.

A stock solution of oxalic acid was prepared in THF (450.2 μL of oxalicacid in 4549.8 μL THF). Separately, 20 mg of the A2-73 freebase wasweighed into a 1.5 mL HPLC vial, to which 300 μL of ethanol was added,along with 74.6 μL of acid stock solution (1.05 equivalents of acid).The sample was temperature-cycled between ambient and 40° C. in 4-hourcycles for 72 hours. The sample was filtered and approximately 100 μL ofthe filtrate was transferred into a 1.5 mL HPLC vial. A 100 μL aliquotof tert-methyl ether was added until precipitation was observed. XRPDdata was collected for the precipitate.

Example 16 Preparation of Dihydrogen Phosphate Form I

A2-73 dihydrogen phosphate Form I was obtained via addition ofphosphoric acid (in THF) to a solution of A2-73 freebase in THF,ethanol, acetonitrile, acetone, 2-propanol or 2-ethoxyethanol.

Specific examples of A2-73 dihydrogen phosphate Form I preparationfollow:

(i) Example 1.

Approximately 300 mg of A2-73 freebase was weighted into a 20 mLscintillation vial, after which, 4 mL of acetone was added to the vial.The apparent pH of the resulting solution was determined using a pHmeter. Phosphoric acid stock solution (1119.4 μL of a solutioncontaining 7.31 mg of phosphoric acid per 74.6 μL of acetone) was added(1.05 equivalents) to the vial, followed by agitation. The apparent pHof the solution was once again measured. The sample was thentemperature-cycled between ambient and 40° C. for ca. 24 hours, withprecipitated solid isolated at the end of ca. 24 hours and air dried forca. 72 hours, followed by characterization by XRPD.

(ii) Example 2.

A stock solution of phosphoric acid was prepared in THF (490 μL ofphosphoric acid in 4510 μL THF). Separately, 20 mg of the A2-73 freebasewas weighed into 1.5 mL HPLC vials, to which 300 μL of the appropriatesolvent (THF, ethanol, acetonitrile, acetone, 2-propanol or2-ethoxyethanol) was added, along with 74.6 μL of acid stock solution(1.05 equivalents of acid). The sample was temperature-cycled betweenambient and 40° C. in 4-hour cycles for 72 hours, and the resultingprecipitate was analyzed by XRPD.

Example 17 Preparation of Edisylate Form I

A2-73 edisylate Form I was obtained via addition of 1,2-ethanedisulfonicacid (in THF) to A2-73 freebase in ethanol, acetonitrile, acetone,2-propanol or 2-ethoxyethanol. Specific examples of A2-73 edisylate FormI preparation follow:

(i) Example 1.

A stock solution of 1,2-ethanedisulfonic was prepared in THF (1170.7 μLof 1,2-ethanedisulfonic acid in 3829.3 μL THF). Separately, 20 mg of theA2-73 freebase was weighed into 1.5 mL HPLC vials, to which 300 μL ofthe appropriate solvent (ethanol, acetonitrile, acetone, 2-propanol, or2-ethoxyethanol) was added, along with 74.6 μL of acid stock solution(1.05 equivalents of acid). The samples were temperature-cycled betweenambient and 40° C. in 4-hour cycles for 72 hours. The samples werefiltered and approximately 100 μL of each filtrate was added to 2 mLglass vials. The vials were left uncapped in a cupboard to allowevaporation. Observed solid post-evaporation was analyzed by XRPD.

(ii) Example 2.

A stock solution of 1,2-ethanedisulfonic was prepared in THF (1170.7 μLof 1,2-ethanedisulfonic acid in 3829.3 μL THF). Separately, 20 mg of theA2-73 freebase was weighed into 1.5 mL HPLC vials, to which 300 μL ofthe appropriate solvent (ethanol, acetone or 2-propanol) was added,along with 74.6 μL of acid stock solution (1.05 equivalents of acid).The samples were temperature-cycled between ambient and 40° C. in 4-hourcycles for 72 hours. The samples were filtered and approximately 100 μLof each filtrate was measured into a 1.5 mL HPLC vials. The vials werecapped and placed in a fridge at ca. 5° C. for ca. 24 hours. The sampleswere checked periodically and observed solids were analyzed by XRPD.Samples which appeared as solutions were placed in a freezer at ca. −20°C. for ca. 24 hours. The samples were checked periodically and observedsolids were analyzed by XRPD.

Example 18 Preparation of Benzoate Form I

A2-73 benzoate Form I was obtained via addition of benzoic acid (in THF)to A2-73 freebase in THF, acetonitrile or acetone. Specific example ofA2-73 benzoate Form I preparation follows:

(i) Example 1.

A stock solution of benzoic acid was prepared in THF (610.6 μL ofbenzoic acid in 4389.4 μL THF). Separately, 20 mg of the A2-73 freebasewas weighed into 1.5 mL HPLC vials, to which 300 μL of the appropriatesolvent (THF, acetonitrile or acetone) was added, along with 74.6 μL ofacid stock solution (1.05 equivalents of acid). The samples weretemperature-cycled between ambient and 40° C. in 4-hour cycles for 72hours. The samples were filtered and approximately 100 μL of eachfiltrate was added to 2 mL glass vials. The vials were left uncapped ina cupboard to allow evaporation. Observed solid post-evaporation wasanalyzed by XRPD.

Example 19 Preparation of Hydrogen Fumarate Form I

A2-73 fumarate Form I was obtained via addition of fumaric acid (in THF)to A2-73 freebase in ethanol or THF. Specific example of A2-73 fumarateForm I preparation follows:

(i) Example 1.

Approximately 200 mg of A2-73 freebase was weighted into a 20 mLscintillation vial, after which, 4 mL of ethanol was added to the vial.The apparent pH of the resulting solution was determined using a pHmeter. Fumaric acid stock solution (1119.4 μL of a solution containing8.66 mg of fumaric acid per 74.6 μL of ethanol) was added (1.05equivalents) to the vial, followed by agitation. The apparent pH of thesolution was once again measured. The sample was then temperature-cycledbetween ambient and 40° C. for ca. 24 hours. As no solid was observed atthe end of ca. 24 hours, the sample was left uncapped in a fume hood andallowed to air-evaporate for ca. 72 hours, followed by characterizationby XRPD.

A stock solution of fumaric acid was prepared in THF (580.3 μL offumaric acid in 4419.7 μL THF). Separately, 20 mg of the A2-73 freebasewas weighed into a 1.5 mL HPLC vial, to which 300 μL of THF was added,along with 74.6 μL of acid stock solution (1.05 equivalents of acid).The sample was temperature-cycled between ambient and 40° C. in 4-hourcycles for 72 hours. The sample was filtered and approximately 100 μLwas transferred to a 2 mL glass vial. The vial was left uncapped in acupboard to allow evaporation. Observed solid post-evaporation wasanalyzed by XRPD.

A stock solution of fumaric acid was prepared in THF (580.3 μL offumaric acid in 4419.7 μL THF). Separately, 20 mg of the A2-73 freebasewas weighed into a 1.5 mL HPLC vial, to which 300 μL of THF was added,along with 74.6 μL of acid stock solution (1.05 equivalents of acid).The sample was temperature-cycled between ambient and 40° C. in 4-hourcycles for 72 hours. The sample was filtered and approximately 100 μL ofthe filtrate was transferred into a 1.5 mL HPLC vial. A 100 μL aliquotof tert-butyl methyl ether was added until precipitation was observed,and the resulting precipitate was analyzed by XRPD.

Example 20 Preparation of Fumarate Form II

A2-73 fumarate Form II was obtained via addition of fumaric acid (inTHF) to A2-73 freebase in ethanol. Specific example of A2-73 fumarateForm II preparation follows:

(i) Example 1.

A stock solution of fumaric acid was prepared in THF (580.3 μL offumaric acid in 4419.7 μL THF). Separately, 20 mg of the A2-73 freebasewas weighed into a 1.5 mL HPLC vial, to which 300 μL of ethanol wasadded, along with 74.6 μL of acid stock solution (1.05 eq of acid). Thesample was temperature-cycled between ambient and 40° C. in 4-hourcycles for 72 hours, and the resulting precipitate was analyzed by XRPD.

Example 21 Preparation of Hydrogen Fumarate Form III

A2-73 hydrogen fumarate Form III was obtained via addition of fumaricacid (in THF) to A2-73 freebase in IPA. Specific example of A2-73hydrogen fumarate Form III preparation follows:

(i) Example 1.

Approximately 300 mg of A2-73 freebase was weighted into a 20 mLscintillation vial, after which, 4 mL of IPA was added to the vial. Theapparent pH of the resulting solution was determined using a pH meter.Fumaric acid stock solution (1119.4 μL of a 1M fumaric acid stocksolution in IPA) was added (1.05 equivalents) to the vial, followed byagitation. The apparent pH of the solution was once again measured. Thesample was then temperature-cycled between ambient and 40° C. for ca. 24hours, with precipitated solid isolated via centrifugation at the end ofca. 24 hours. The isolated solid was air-dried for ca. 72 hours,followed by characterization by XRPD.

A stock solution of fumaric acid was prepared in THF (580.3 μL offumaric acid in 4419.7 μL THF). Separately, 20 mg of the A2-73 freebasewas weighed into a 1.5 mL HPLC vial, to which 300 μL of IPA was added,along with 74.6 μL of acid stock solution (1.05 equivalents of acid).The sample was temperature-cycled between ambient and 40° C. in 4-hourcycles for 72 hours, and the resulting precipitate was analyzed by XRPD.

Example 22 Preparation of Fumarate Form IV

A2-73 fumarate Form IV was obtained via addition of fumaric acid (inTHF) to A2-73 freebase in 2-ethoxyethanol. Specific example of A2-73fumarate Form IV preparation follows:

(i) Example 1.

A stock solution of fumaric acid was prepared in THF (580.3 μL offumaric acid in 4419.7 μL THF). Separately, 20 mg of the A2-73 freebasewas weighed into a 1.5 mL HPLC vial, to which 300 μL of 2-ethoxyethanolwas added, along with 74.6 μL of acid stock solution (1.05 equivalentsof acid). The sample was temperature-cycled between ambient and 40° C.in 4-hour cycles for 72 hours. The sample was filtered and approximately100 μL was transferred to a 2 mL glass vial. The vial was left uncappedin a cupboard to allow evaporation. Observed solid post-evaporation wasanalyzed by XRPD.

Example 23 Anavex2-73 Fumarate Form V

A2-73 fumarate Form V was obtained via addition of fumaric acid (in THF)to A2-73 freebase in IPA. Specific example of A2-73 fumarate Form Vpreparation follows:

(i) Example 1.

Approximately 300 mg of A2-73 freebase was weighted into a 20 mLscintillation vial, after which, 4 mL of IPA was added to the vial. Theapparent pH of the resulting solution was determined using a pH meter.Fumaric acid stock solution (1119.4 μL of a 1M fumaric acid stocksolution in IPA) was added (1.05 equivalents) to the vial, followed byagitation. The apparent pH of the solution was once again measured. Thesample was then temperature-cycled between ambient and 40° C. for ca. 24hours, with precipitated solid isolated via centrifugation at the end ofca. 24 hours. The isolated solid was air dried for ca. 72 hours,followed by characterization by XRPD.

Example 24 Transdermal Patch

A 63 year-old male presents with early signs of Alzheimer's disease. Heis administered a pharmaceutical composition via transdermal patchcontaining 100 mg ANAVEX2-73 freebase with a 4 cm2 patch replacedapproximately every three days administration for 120 days. Blood levelsof about 10 ng/ml are being maintained. His cognitive functionstabilizes during that period, with no additional loss detected.

Example 25 Transdermal Patch

A 57 year-old female presents with signs of early onset Alzheimer'sdisease. She is administered via a 9 cm2 transdermal patch containing apharmaceutical composition containing 200 mg ANAVEX2-73 freebase withthe patch replaced weekly for 180 days. Blood levels of about 12 ng/mlare being maintained. Her cognitive function stabilizes during thatperiod with no additional loss detected.

Example 26 Extended Released Oral Dosage Form

An 84 year old male with unspecified progressive dementia isadministered 30 mg of ANAVEX2-73 fumarate in an enteric coated tabletevery other day for 180 days. Blood levels reveal about 25 mg per dayare being administered. His cognitive function stabilizes during thatperiod with no additional loss detected.

Example 27 Extended Released Oral Dosage Form

An 77 year old female with unspecified progressive dementia isadministered 50 mg of ANAVEX2-73 freebase in an enteric coated tabletevery other day for 180 days. Blood levels reveal about 20 mg per dayare being administered. Hers cognitive function stabilizes during thatperiod with no additional loss detected.

Example 28 Sig1-R Agonist ANAVEX2-73 Enhances Autophagic Activity

To study the effect of ANAVEX2-73 on autophagy, human HeLa cells weretreated with the compound and autophagic activity was analyzed byinvestigating the flux of LC3-II. LC3-II is the lipidated form of LC3,which (partially) stays attached to autophagosomes and thus, getsdegraded by lysosomes. Therefore, the quantification of the LC3-II flux,using Bafilomycin A₁ (BafiA₁; 2 μM) for inhibition of lysosomaldegradation, directly corresponds to cellular autophagic activity. Asdisplayed in FIG. 35, ANAVEX2-73 significantly induced autophagic fluxwhen compared to control conditions. There is a concentration-dependentand significant increase in the autophagic-flux following application ofANAVEX2-73: an increase of over 2-fold at 10 μM and over 1.5-fold at 1μM ANAVEX2-73 (FIG. 35A). As standard positive control to provoke theinduction of autophagy, HeLa cells were incubated with EBSS, whichresembles nutrient deprivation as autophagy stimulus.

ANAVEX2-73 and other known experimental Sig-1R agonists were used in theexperiments. Such compounds include (+)-pentazocine, (+)-SKF10,047,SA4503 (14243,4-dimethoxyphenyDethyl]-4-(3-phenylpropyl)piperazine), andPRE-084 (2-morpholin-4-ylethyl 1-phenylcyclohexane-1-carboxylate). Incontrast to ANAVEX2-73, PRE-084 and the other experimental compounds arenot applicable in clinical studies for various reasons. However, sincethe Sig-1R ligand PRE-084 exhibits activities in the central nervoussystem in animal models such as nootropic and antidepressant activities,this compound was included in some of the flux assays as control. It wasfound that PRE-084 also promotes autophagic flux in HeLa cells (FIG.35B); PRE-084 induces the autophagic flux at over 1.5 fold at 1 μM,which was comparable with ANAVEX2-73 at the same concentration (FIG.35B).

Next, the Western blot experiments were complemented by directvisualization of the extent of autophagosome appearance in HEK293 cells.To do so, ANAVEX2-73 (1 μM) was applied to HEK293 cells stablyexpressing a GFP-LC3B reporter construct. This cell model allows directmonitoring of the accumulation of LC3-II-positive autophagosomalstructures upon BafiA₁ supplementation by confocal fluorescencemicroscopy. Indeed, ANAVEX2-73 treatment resulted in an overallincreased number of LC3-II-positive puncta and autophagic flux (FIG.35C).

Taken together, in both independent cell assays and in two differenthuman cell lines, Sig-1R activation induced a significantly increasedautophagic flux. Part of the effect of ANAVEX2-73 as Sig-1R ligand couldpotentially be ascribed to its effects at the muscarinic ACh-receptor.But not much is known about the impact of mACh receptors on autophagy.In fact, so far there is only one report in the literature showing thatACh-induced autophagy has cytoprotective effects through the muscarinicACh-receptor activated-AMPK-mTOR pathway. On the other hand, our findingthat also PRE-084, as an exclusive selective Sig-1R agonist, wasinducing autophagic flux, strongly supports ANAVEX2-73's effects onautophagy as being mediated by Sig-1R activation. Moreover, noexperimental data exist that an activation of the muscarinicACh-receptor has beneficial effects on protein aggregation andproteostasis, as clearly ANAVEX2-73 has, as shown in Example 30 below.

Example 29 Sig-1R Activation Induces ULK1 Phosphorylation and AffectsExpression Levels of Distinct Autophagy Network Factors

Activation of the serine/threonine protein kinase ULK1 (unc-51-likekinase 1) via phosphorylation at serine 555 indicates stimulation of thecanonical autophagy pathway. ANAVEX2-73 significantly induced ULK1serine 555 phosphorylation (up to 2-fold at 1 μM; FIG. 36A). PRE-084 wasalso analyzed as Sig-1R agonist and it was found that it similarlypromotes ULK1 serine 555 phosphorylation (up to 1.5 fold at 1 μM FIG.36B). It has to be mentioned that this activating ULK1 phosphorylationcan be inhibited by mTOR as well as stimulated via AMPK kinase. Both arebasal physiological sensors of nutritional conditions and key signaltransducers of canonical autophagy stimulation. ULK1 is in fact thesignal mediating the induction of the formation of the phagophore duringthe autophagy process and, therefore, a central promoter of autophagy.ULK1 itself functions in a complex with at least three protein partners:FIP200 (focal adhesion kinase family interacting protein of 200 kDa),ATG (autophagy-related protein) 13 (ATG13), and ATG101. The fact that acomplex pattern of upstream pathways (including mTOR and AMPK) convergeon ULK1, suggests that this complex acts as a node, converting multiplesignals into autophagosome formation.

In view of the results found by the inventors that Sig-1R activationsignificantly induces ULK1 phosphorylation and autophagic flux, therelative expression levels of key autophagy network factors representingdifferent set points in the autophagy process was next investigatedafter treatment of HeLa cells with ANAVEX2-73 employing a PCR autophagyarray (FIG. 36C). Most prominently, an ANAVEX2-73-mediated induction ofthe mRNA expression of GABA Type A Receptor Associated Protein Like 1(GABARAPL1; expression level of approx. 2.7; cut-off for induction wasset at the expression level of 1.5) was found, which, like GABARAP,associates with autophagic vesicles and is involved in the autophagyprocess.

GABARAPL1 belongs to the human MAP1LC3 family consisting of six ATG8orthologs, MAP1LC3A, MAP1LC3B, MAP1LC3C and three MAP1LC3 paralogs, theGABA receptor-associated proteins GABARAP1, GABARAPL1 and GABARAPL2 withpartially redundant roles in autophagy. In addition, the expression ofthe ubiquitin and autophagy receptor SQSTM1/p62 involved in selectivemacroautophagy pathways was enhanced by ANAVEX2-73 (expression level ofapprox. 2.9). Moreover, there was also a clear tendency towards theinduction of ATG12, which is conjugated to ATG5 and is building anautophagosomal protein complex that finally acts together with ATG16L1in autophagosomal biogenesis. Consistently, the expression of ATG16L1appeared also enhanced following treatment of the cells with ANAVEX2-73(FIG. 36C). Moreover, it is clear that none of the autophagy networkfactors included in this PCR array was downregulated in its expressionupon treatment with ANAVEX2-27, supporting the key finding that Sig-1Ractivation has a positive modulatory effect on autophagy.

Example 30 ANAVEX2-73 Positively Regulates Autophagy, IncreasesProteostasis Capacity and Improves Protein Aggregation-MediatedParalysis in C. elegans

Autophagy modulation by ANAVEX2-73 in vitro and its impact on some keyautophagy network factors prompted the inventors to further analyze theimpact of Sig-1R activation by ANAVEX2-73 on autophagy and proteostasisalso in vivo, employing the C. elegans model. The nematode ortholog ofthe human Sig-1R is W08F4.3 and is expressed in several tissues,including the muscular system. To monitor autophagic flux in vivo theinventors employed a GFP-LGG-1 reporter worm strain. LGG-1 is a nematodeortholog of the mammalian GABARAP, and the GFP-tagged protein can beused to evaluate autophagic activity by Western blotting as well asconfocal fluorescence microscopy. Employing Western blotting, the levelsof GFP-LGG-1-II plus BafiA₁ and without BafiA₁ were analyzed,analogously to the flux measurements in HeLa cells as shown in FIG. 37.Indeed, ANAVEX2-73 (80 μM) significantly enhanced autophagic flux in C.elegans almost 2-fold (Nematodes treated with BafiA₁ or DMSO for 6 h.FIG. 37A).

To further substantiate this finding we used confocal fluorescencemicroscopy to directly visualize autophagosomal structures, as indicatedby GFP-LGG-1-positive puncta. ANAVEX2-73 supplementation (plus/minusBafiA1) significantly increased the number of GFP-LGG1 puncta, which isindicative of increased autophagic activity; treatment of worms withANAVEX2-73 lead to a relative increase in numbers of puncta after BafiA1treatment when compared to control worms. In fact, a significantincrease was found; autophagic flux as observed in vivo is induced byANAVEX2-73 by approx. 2.5-fold (FIG. 37B; The number of GFP-positiveautophagosomal structures (indicated by arrowheads) were counted inthree independent experiments and in each experiment in at least 8-11respective head regions of worms), which is consistent with the Westernblot analysis (FIG. 37A).

Taken together, the in vitro and in vivo data clearly show that theSig-1R agonist ANAVEX2-73 induces autophagy, as indicated by autophagicflux measurements. This encouraged the inventors to further look intothe functional consequences of autophagy induction, focusing on theimpact of the degradative pathway on proteostasis in vivo. Therefore,human Aβ42-expressing worms characterized by a time-dependent paralysiswere employed, due to the accumulation of Aβ42 oligomers and highmolecular weight aggregates in body wall muscle cells; it is stressedhere that Aβ42-expressing worms are not considered as a model for AD,but rather as an experimental model for general proteostasis stress andproteotoxicity, where protein aggregation in muscle cells leads do aclear-cut phenotype (here, paralysis). Aβ42 protein aggregates werestained in situ with thioflavine. Compared to control worms, treatmentof Aβ42-worms with ANAVEX2-73 reduced the number of thioflavine-positiveAβ42 aggregates (FIG. 38A; worms were treated with 80 μM ANAVEX2-73 orM9 medium (control), for nine consecutive days), suggesting that theinduction of autophagy impacts on proteostasis, presumably by anenhanced clearance of Aβ42 aggregates, resulting in a reduced tissuedeposition of aggregates. The accumulation of Aβ42 aggregates in themuscle cells is known to lead to an enhanced paralysis of the worms overtime. To analyze the impact of ANAVEX2-73-induced autophagy on thetime-dependent movement behavior, the extent of this paralysis wasinvestigated. C. elegans were treated with the compound (or M9 buffer ascontrol) up to 12 days and paralysis was quantified daily. Employing twoconcentrations of ANAVEX2-73 (50 and 100 μM), we found a clear reductionin paralysis in the two ANAVEX2-73 treatment groups; these groupsclearly separate from the controls with respect to the extent ofparalysis (FIG. 38B; Worms were maintained in the presence of ANAVEX2-73or M9 buffer and the paralysis phenotype was examined daily.) Theparalyzed fraction is significantly different comparing ANAVEX2-73treated and control worms. Therefore, ANAVEX2-73 clearly decelerates theparalysis rate and counteracts the time-dependent movement impairment inAβ42-expressing worms.

The findings described herein, that autophagy induction via a Sig-1Ragonist is directly impacting on proteostasis by reducing proteinaggregation and proteotoxicity-induced behavioral impairment in wormsdemonstrates a role of Sig-1R activation in the prevention and treatmentof neurodegeneration associated with an imbalanced protein homeostasis.Consistently with the ANAVEX2-73-induced increase in proteostasiscapacity observed herein, the involvement of Sig-1R deficiency ordysfunction has been described in ALS, a disorder with a highlydisturbed protein homeostasis and characteristic intracellular proteinaggregation. For instance, it has been shown that (1) Sig-1R missensemutation can cause ALS, (2) the knock-out of Sig-1R accelerates diseasein SOD1-mutant mice, and (3) an ALS-linked mutant Sig-1R causesaccumulation of autophagic material and reduced autophagy. Furthermore,in support of a protective role of Sig-1R activity, it was previouslydescribed that (1) treatment with the experimental drug PRE-084 improvesSOD1 mice pathology, (2) mutant Sig-1R expression induces cytosolicALS-linked TDP43 and FUS accumulation in cells, and (3) PRE-084 improvesmotor function and motor neuron survival in ALS mice. Fully consistentwith the finding herein, the overexpression of Sig-1A receptor increasesthe number of p62/SQSTM1 and LC3B puncta indicative of autophagyactivation in human disease tissue.

Several steps of the autophagic processes are amenable to therapeuticmodulation and different autophagy-activating compounds have alreadybeen studied at various experimental levels (in vitro and in vivo) andmodels of human diseases, including cancer and neurodegeneration.Regarding an effective intervention of neurodegenerative disorders, ofcourse, for any compound planned to be studied in humans in the contextof the central nervous system, besides toxicity and safety issues, alsothe permeability of the blood-brain barrier has to be secured. Oneexample of a compound targeting autophagy is lithium, which is in usefor the treatment of bipolar disorders and is also an activator ofautophagy, by interfering with upstream steps in autophagy induction.Metformin and simvastatin have also been shown, experimentally, topromote autophagy, both supposedly via the activation of AMPK, and areused for the treatment of diabetes and obesity, respectively. Sig-1Ragonists are under intense investigation for the treatment of differentneurodegenerative diseases, including AD and ALS. Without beingconstrained by theory, it is the combination of receptor activities thatmay make ANAVEX2-73 an interesting compound for AD therapy.

Taken together, the results presented in Examples 28-30 herein show thatSig-1R activation (a) enhances the autophagic flux in human cells and inC. elegans, and (b) has positive effects on proteostasis. A novelactivity of the compound ANAVEX2-73 having dual selectiveSig-1R/muscarinic activities in neurons is described. The activity ofthis drug comprises a potent induction of autophagy, in vitro and invivo, leading to an increased proteostasis capacity, and even tobeneficial effects on the time-dependent paralysis phenotype inAβ42-expressing C. elegans. A specific induction of the autophagyprocess and a subsequent stabilization of the proteostasis in neuronsrepresents one important step towards the stabilization of neuronalsurvival and function, and can help to prevent age-associatedneurodegeneration.

Introduction for Examples 28-30

The pathogenesis of neurodegenerative disorders, including Alzheimer'sand Parkinson's disease (AD, PD) as well as Amyotrophic LateralSclerosis (ALS), has been linked to a disturbed protein homeostasis.Therefore, the control and maintenance of proteome integrity andproteostasis is of utmost importance. Cellular proteostasis includesprotein folding, protein assembly, refolding of damaged proteins as wellas protein degradation and is under the control of a fine tuned networkof factors including chaperones such as heat shock protein 70 (HSP70)and distinct co-chaperones. For intact function and long-term survivalof the cell, it is crucial to remove misfolded proteins via specializedprocesses; the two major cellular degradation pathways are ubiquitinproteasome system (UPS) and autophagy. The UPS is of particularimportance for the physiological protein turnover but is limited in thedegradation substrates and the autophagic-lysosomal pathway isresponsible for the clearance of aggregated and disease-associatedproteins, especially under pathogenic and aging conditions.

Autophagy is a highly dynamic vesicle-mediated cellular degradationpathway involving double-membraned vesicles, called autophagosomes,which sequester large protein complexes (protein aggregates), and evenwhole organelles and deliver them to lysosomes for degradation. Underlow nutrition and energy conditions autophagy guarantees energy supplyby generating amino acid building blocks via recycling. In addition,autophagy plays an important role as a stress and adaptive response andrescue mechanism to maintain cell survival and function. Canonicalautophagy responds to environmental cues via a variety of factors thatmainly belong to homologs of autophagy-related (atg) genes originallyidentified in yeast. The mammalian target of rapamycin (mTOR) complex 1(mTORC1) negatively regulates autophagic activity via inhibitoryphosphorylation of ULK1 and is the key initial regulator of canonicalautophagy. More downstream membrane expansion is modulated by twoubiquitin-like conjugating systems (ATG12-ATG5 and ATG8/LC3) and theATG18 protein family members of WD repeat domain phosphoinositideinteracting 1-3 (WIPI1-3).

There is a great amount of data linking dysfunction and malfunction ofautophagy to neurodegenerative disease and consistent with its role inproteostasis, to the accumulation of protein aggregates. Thus, themodulation of autophagy has become one key pharmacological target inneurodegeneration. In fact, there are multiple overlaps of autophagy andpathogenesis pathways in AD, PD and ALS. Recently different alternativeviews and new pharmacological targets towards AD prevention andtreatment are evolving and include a strong focus on the autophagyprocess.

There are two subtypes of sigma receptors, sigma-1 and sigma-2, bothhighly expressed in the central nervous system. Sigma-1 receptor(Sig-1R) was cloned in 1996 and represents an integral membrane proteinof 223 amino acids protein localized to the endoplasmic reticulum (ER)(and the ER-mitochondrial interface) suggesting a role as ER chaperone.Sig-1R was shown to promote cellular survival by (1) ensuring Ca2+signaling from the ER into mitochondria, (2) enhancing the signaling ofER to the nucleus, and (3) attenuating free radical damage by modulationof the activity of Nrf2, a redox-responsive transcription factor.Structurally, Sig-1R ligand binding is characterized and the crystalstructure of the human receptor is solved.

In general, deficits in Sig-1R expression or activity are linked toneurodegeneration and the activation of Sig-1R is associated withneuroprotection in different in vitro and in vivo models, employingdifferent types of pharmacological Sig-1R activators with differentpharmacological profiles. The pharmacological activation of Sig-1R leadsto pluripotent modulatory downstream effects and incorrect function ofSig-1R is strongly suggested to be also involved in the pathogenesis ofneurodegeneration. This is the basis of an effort to design novel andhighly specific pharmacological Sig-1R activators for the therapy ofneurodegenerative disease, including.

In this context a novel Sig-1R agonist,tetrahydro-N,N-dimethyl-2,2-diphenyl-3-furanmethanamine hydrochloride(ANAVEX2-73), was developed. Pharmacologically ANAVEX2-73 shows a mixedactivity on Sig-1R as well as muscarinic receptor, acting with describedaffinities in the low micromolar range. Previously, pre-clinical studiesin animal models demonstrated robust disease-modifying activities ofANAVEX2-73. Regarding AD, ANAVEX2-73 has undergone testing in Phase 2atrial of patients demonstrating a favorable safety profile and aconcentration-dependent improvement against exploratory endpoints. Avariety of neuromodulatory and neuroprotective effects are also alreadyknown for ANAVEX2-73 including mitochondrial protection in mouse modelsof AD, regulation of ERK activation and promotion of survival ofastrocytes, as well as protection against oxidative stress.

First evidence for a possible link of Sig-1R, autophagy andneurodegeneration have been recently shown in the context of ALS. It wasdiscovered that ALS-linked mutant Sig1-R causes an accumulation ofautophagic material and actually reduced autophagy. In addition, it wasfound that a small-molecule Sig-1R modulator induces autophagicdegradation of programmed-death ligand 1 (PD-L1) in cancer cells. Thesefindings prompted us to study the potential of ANAVEX2-73 to effectautophagy in human HeLa and HEK293 cells (in vitro) as well as in C.elegans (in vivo), employing standard measures to analyze autophagicactivity, which are well-established by the inventors. Moreover, theeffects of ANAVEX2-73 on protein aggregation and subsequently the impactof protein aggregates on movement behavior in C. elegans were studied.Excitingly, ANAVEX2-73 is a potent inducer of autophagic flux in vitroand in vivo and ameliorates protein aggregate formation and paralysis inC. elegans.

Materials and Methods for Examples 28-30

Cell culture and microscopy. HeLa and HEK293A cells were cultured inDMEM (Invitrogen, Carlsbad, Calif., USA, 41965062) supplemented withactive FBS (Life Technologies GmbH, Carlsbad, Calif., USA, 10270106), 1×ABAM (Invitrogen, 15240-062) and 1 mM sodium pyruvate (Invitrogen,1136-088). After medium change, the cells were treated for 2 h with 10,1, and 0.1 μM ANAVEX2-73 and PRE-084 (Tocris, Bristol, UK, 0589),respectively; ANAVEX2-73 was provided by ANAVEX Life Sciences Corp, NewYork, N.Y., USA. Afterwards Bafilomycin A₁ (Bafi.A_(b;) 2 μM) (TorontoResearch Chemicals, North York, ON, Canada, B110000) or DMSO was addedfor a further 2 h and the cells were eventually harvested. Western blotanalyses were performed as described previously [40, 41]. Briefly, cellswere subjected to SDS-PAGE using precast NuPAGE 4%-12% Bis-Tris gels(Invitrogen, NP0322). Proteins were detected by chemiluminescence usingthe Amersham Imager 600 (GE).

Confocal fluorescence microscopical analyses of HEK293A cells stablyexpressing GFP-LC3B were performed with the laser scanning microscopeLSM 710 (Zeiss, Oberkochen).

C. elegans strains, maintenance, methods. C. elegans were maintainedaccording to standard procedures on nematode growth medium (NGM) platesseeded with HB101 E. coli. The following strains were employed in thisstudy: GFP::LGG-1 (ex[PIggI::IggI::GFP]/pRF4; maintained at 20° C.; andthe strain CL2006 (dvls2 [pCL12(unc-54/human Aβ peptide 1-42)+pRF4]),maintained at 15° C.

For analysis of paralysis rate, synchronous CL2006 nematodes werecultivated at 15° C. on plates seeded with HB101 E. coli re-suspended inM9 (control) or 100 μM and 50 μM ANAVEX2-73. Starting at first day ofadulthood, worms were transferred to fresh plates daily and were testedfor paralysis by tapping their nose with a platinum wire. Worms thatmoved their nose but failed to move their bodies were scored asparalyzed. Dead worms or worms showing other phenotypes were notincluded into the statistics. Staining of amyloid β42 aggregates usingthioflavine S (Sigma T1892) were carried out as previously described.Worms were mounted on 2% agar pads on a glass slide and confocalfluorescence microscopical analyses were performed with the LSM 710(Zeiss, Oberkochen) laser scanning microscope.

For analysis of autophagic activity, synchronous nematodes expressingGFP::LGG-1 were cultivated at 20° C. At first day of adulthood, wormswere transferred to 80 μM ANAVEX2-73 or control M9 liquid culture mediumfor 2 h and subsequently were treated with Bafilomycin A1 or DMSO(control) for 4-6 h. Thereafter worms were lysed for Western blotting oranalyzed by confocal fluorescence microscopy.

Western blot analyses were performed as described previously. Generally,12 worms were subjected to SDS-PAGE using precast NuPAGE 4-12% Bis-Trisgels (Invitrogen, NP0322). Proteins were detected by chemiluminescenceusing the Fuji LAS-3000 dark box (Fujifilm, Dusseldorf).

1. A crystalline form oftetrahydro-N,N-dimethyl-2,2-diphenyl-3-furanmethanamine (A2-73), whereinthe crystalline form is a fumarate salt or a freebase. 2-7. (canceled)8. The crystalline form of claim 1, wherein the fumarate salt ischaracterized by the XRPD pattern shown in FIG. 29, FIG. 30, FIG. 32,FIG. 33, and FIG.
 34. 9. The crystalline form of claim 8, wherein thefumarate salt characterized by the XRPD pattern shown in FIG. 29 isfurther characterized by the particle shapes depicted in FIG. 28, andthe fumarate salt characterized by the XRPD pattern shown in FIG. 32 isfurther characterized by the particle shapes depicted in FIG.
 31. 10.(canceled)
 11. The crystalline form of claim 1, wherein the freebase ischaracterized by the XRPD pattern shown in FIG.
 16. 12. The crystallineform of claim 11, wherein the crystalline form characterized by the XRPDpattern shown in FIG. 16 is further characterized by the particle shapesdepicted in FIG.
 15. 13. A dosage form comprising a therapeuticallyeffective amount of A2-73 in a crystalline form selected from the groupconsisting of A2-73 freebase and A2-73 fumarate salt.
 14. The dosageform of claim 13, wherein the dosage form comprises from about 1 mg toabout 50 g, from about 1 mg to about 500 mg, or about 1 mg to about 100mg of A2-73 freebase or A2-73 fumarate salt.
 15. The dosage form ofclaim 13, wherein the dosage form is formulated for extended release ofcrystalline freebase or fumarate salt of A2-73.
 16. (canceled)
 17. Thedosage form of claim 15, wherein the dosage form comprises from about 1mg to about 500 mg of A2-73 freebase.
 18. The dosage form of claim 16,wherein the dosage form is a transdermal patch.
 19. The dosage form ofclaim 18, wherein the transdermal patch comprises from about 40 mg toabout 60 mg, from about 80 mg to about 120 mg, or from about 180 mg toabout 220 mg of A2-73 freebase.
 20. The dosage form of claim 16, whereinthe dosage form is an enteric coated oral formulation.
 21. The dosageform of claim 20, wherein the enteric coated oral formulation comprisesfrom about 1 mg to about 50 mg A2-73 freebase. 22-24. (canceled)
 25. Thedosage form of claim 24, wherein the dosage form is a transdermal patch.26. The dosage form of claim 25, wherein the transdermal patch comprisesfrom about 1 mg to about 55 mg of A2-73 fumarate salt.
 27. The dosageform of claim 24, wherein the dosage form is an enteric coated oralformulation.
 28. The dosage form of claim 27, wherein the enteric coatedoral formulation comprises from about 10 mg to about 50 mg, from about20 mg to about 30 mg, or from about 15 mg to about 25 mg of A2-73fumarate salt.
 29. A pharmaceutical formulation for delivery of A2-73,the formulation comprising a therapeutically effective amount of thecrystalline form of A2-73 selected from A2-73 freebase and A2-73fumarate salt.
 30. The formulation of claim 29, wherein the formulationfurther comprises one or more pharmaceutically acceptable excipientsselected from chemical enhancers, humectants, pressure sensitiveadhesives, antioxidants, solubilizers, thickening agents, plasticizers,adjuvants, carriers, excipients, vehicles, and any combinations thereof.31. The formulation of claim 30, wherein the one or more excipients areselected for oral, transdermal, parenteral, intraperitoneal,intravascular, subcutaneous, by inhalation spray, rectal, orintrapulmonary administration.
 32. (canceled)
 33. The formulation ofclaim 29, wherein the formulation is an oral formulation comprising fromabout 1% to about 100% by weight crystalline freebase or fumarate saltof A2-73.
 34. The formulation of claim 29, wherein the formulation isfor extended delivery of crystalline freebase or fumarate salt of A2-73.35. The formulation of claim 34, wherein the formulation comprises fromabout 1 mg to about 50 g of crystalline freebase or fumarate salt ofA2-73.
 36. The formulation of claim 34, wherein the formulation is asubcutaneous injectable dosage formulation comprising from about 0.5 gto about 3 g of crystalline freebase or fumarate salt of A2-73.
 37. Theformulation of claim 29, wherein the formulation is a transdermal patch.38. The formulation of claim 37, wherein the patch comprises from about40 mg to about 60 mg, from about 80 mg to about 120 mg, or about 180 mgto about 220 mg of A2-73 freebase.
 39. The formulation of claim 37,wherein the patch comprises from about 1 mg to about 55 mg of A2-73fumarate salt.
 40. The formulation of claim 29, wherein the formulationis an oral formulation.
 41. The formulation of claim 40, wherein theoral formulation comprises from about 1 mg to about 50 mg A2-73freebase.
 42. The formulation of claim 40, wherein the oral formulationcomprises from about 10 mg to about 50 mg, from about 20 mg to about 30mg, or from about 15 mg to about 25 mg of A2-73 fumarate salt. 43.(canceled)
 44. The formulation of claim 29, wherein the formulation is asubcutaneous dosage form comprises from about 0.1 to about 5 g ofcrystalline freebase or fumarate salt of A2-73.
 45. A method ofadministering A2-73 to a subject in need thereof, the method comprisingadministering the A2-73 to the subject in a dosage form comprising acrystalline form of A2-73 selected from A2-73 freebase A2-73 fumaratesalt. 46-48. (canceled)
 49. The method of claim 45, wherein the dosageform is an extended release transdermal patch and wherein thecrystalline A2-73 is administered topically using a transdermal patch.50. The method of claim 49, wherein transdermal patch is replacedweekly.
 51. The method of claim 49, wherein the transdermal patchmaintains a level of A2-73 in the blood of the subject ranging fromabout 5 ng/ml to about 15 ng/ml and particularly about 10 ng/ml ismaintained.
 52. The method of claim 45, wherein the dosage form is anenteric coated oral dosage form and wherein the crystalline freebase orfumarate salt of A2-73 is administered orally using the enteric coatedoral dosage form.
 53. A method of treating Alzheimer's disease in asubject in need thereof, the method comprising administering a dosageform comprising a therapeutically effective amount of a crystalline formof A2-73 selected from A2-73 freebase or a A2-73 fumarate salt.
 54. Amethod of treating a progressive dementia in a subject in need thereof,the method comprising administering a dosage form comprising atherapeutically effective amount of a crystalline form of A2-73 selectedfrom A2-73 freebase or a A2-73 fumarate salt.
 55. A method of treating aneurodegenerative disease in a subject in need thereof, the methodcomprising administering to the subject an anti-neurodegenerativeeffective amount of crystalline A2-73 selected from A2-73 freebase or aA2-73 fumarate salt.
 56. The method of claim 52, wherein thedegenerative disease is selected from Alzheimer's disease, Parkinson'sdisease, prion diseases, Huntington's disease, motor neuron diseases(MND) such as amyotrophic lateral sclerosis, spinocerebellar ataxia(SCA), and spinal muscular atrophy (SMA).
 57. The method of claim 52,wherein the anti-neurodegenerative effective amount of A2-73 is about0.5 mg/day to about 100 mg/day.
 58. The method of claim 52, wherein theanti-neurodegenerative effective amount of A2-73 is about 1 to about 60mg/day
 59. The method of claim 52, wherein the anti-neurodegenerativeeffective amount of A2-73 is about 20 to about 50 mg/day.
 60. The methodof claim 52, wherein the anti-neurodegenerative effective amount ofA2-73 is about 20 to about 30 mg/day.
 61. The method of claim 52,wherein the anti-neurodegenerative effective amount of A2-73 is about 15to about 25 mg/day.
 62. The method of claim 52, wherein theanti-neurodegenerative effective amount of A2-73 provides blood levelsof about 10 ng/ml, about 12 ng/ml, about of A2-73.